Human skin equivalents expressing exogenous polypeptides

ABSTRACT

The present invention relates generally to compositions for wound closure. More specifically, the present invention provides human skin equivalents engineered to express exogenous polypeptides (e.g., antimicrobial polypeptides and keratinocyte growth factor 2) and compositions and methods for making human skin equivalents engineered to express exogenous polypeptides. In addition, the present invention provides methods for treatment of wounds with human skin equivalents engineered to express exogenous polypeptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of pending U.S. patent applicationSer. No. 12/690,160, filed Jan. 20, 2010, which is a continuation ofpending U.S. patent application Ser. No. 10/909,119, filed Jul. 30, 2004(U.S. Pat. No. 7,674,291 which issued on Mar. 9, 2010), which claimspriority to expired U.S. Provisional Patent Application No. 60/491,869,filed Aug. 1, 2003, and U.S. Provisional Patent Application No.60/493,664, filed Aug. 8, 2003, all of which are herein incorporated byreference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under STTR Fast-TrackGrant Phase I No.: N#1 R41 AR 0530349-01 and Phase II #4 R42 AR050349-02 awarded by the National Institutes of Health. The governmenthas certain rights in the invention.

FIELD OF THE INVENTION

The present invention relates generally to compositions for woundclosure. More specifically, the present invention provides human skinequivalents engineered to express exogenous polypeptides (e.g.,antimicrobial polypeptides and keratinocyte growth factor 2) andcompositions and methods for making human skin equivalents engineered toexpress exogenous polypeptides. In addition, the present inventionprovides methods for treatment of wounds with human skin equivalentsengineered to express exogenous polypeptides.

BACKGROUND

Chronic wounds affect three million people each year in the U.S. Chronicwounds generally involve any break, or ulceration, of the skin that isof long duration or recurs frequently. Such wounds cause pain, loss offunction, force changes in an individual's life through potential lackof mobility, take extended periods of time for recovery, and requirehigh amounts of patient compliance for recovery.

Chronic wounds disrupt the integrity of the skin by tearing, cutting,piercing or breaking the tissue. The causes may be structural, such asinjury, or physiological, such as an underlying disease. The mostfrequently occurring skin wounds are venous ulcers, pressure ulcers anddiabetic foot ulcers.

Chronic wounds are a serious health concern with substantial morbidity.They also are a source of frustration to both physician and patient, aslengthy treatments, treatment failures and the need for long periods ofpatient compliance prove challenging. The wounds take so long to healthat compliance drops off and worsens when reversals occur or new ulcersappear.

Chronic wounds occur in individuals with underlying diseases of varioustypes whose medical conditions compromise the body's ability to repairinjured tissue on its own. Despite the use of a variety of medical andsurgical treatments, chronic wounds can take months or even years toheal and frequently recur. These wounds are often large and unsightlyand may be painful in some patients.

Chronic wounds are of three major types: venous stasis ulcers, diabeticulcers and pressure ulcers. A venous ulcer is an ulceration thatdevelops on the ankle or lower leg in patients with chronic vasculardisease. In these patients, blood flow in the lower extremities isimpaired, leading to edema (swelling) and mild redness and scaling ofthe skin that gradually progress to ulceration. Venous ulcers are acondition affecting 500,000-700,000 patients in the US and 1.3 millionpeople in the industrialized world.

A diabetic ulcer is a chronic wound that occurs in patients withdiabetes. While the actual cause of the ulcer in these patients is aninjury such as a callus, blister or foreign body such as a pebble orsplinter, it is the patient's underlying disease that places him or herat high risk for developing an ulcer. Important risk factors include:inadequate local blood supply, which impairs their ability to repairinjured tissue and ward off infection, and reduced sensation in theextremities, which causes the initial injury to go unrecognized until itbecomes a serious, chronic wound. Diabetic ulcers are a conditionaffecting just under 500,000 patients in the US and 1.2 million peoplein the industrialized world.

A pressure ulcer is defined as any lesion caused by unrelieved pressureon tissues that are located over a bony prominence on the body. Pressureulcers were formerly referred to as bedsores or decubitus ulcers.Pressure ulcers develop in immobile patients whose tissues are subjectedto continuous pressure from bones on the interior and hard surfaces suchas beds or chairs on the exterior. In addition to their immobility,patients at risk for the development of pressure ulcers typically havepoor nutritional status, inadequate hydration, and other underlyingmedical conditions that compromise their ability to heal injuries.Pressure ulcers affect over 1.6 million people in the US and 4.1 millionpeople in the industrialized world. Estimates of the prevalence of theseconditions vary greatly. Estimates as high as 12 million patients havebeen reported for all types of chronic wounds in the industrializedmarkets.

Chronic wounds can be of variable sizes and depths. In general, thereare four layers of tissue that can potentially sustain injury in awound, the epidermis, or outermost layer; the dermis; the subcutaneoustissue; and, at the deepest layer, muscle, tendon, and bone.Partial-thickness ulcers involve a loss of skin that is limited to theepidermis and, potentially, part of the dermis. These wounds heal byepithelialization (proliferation and migration of epithelial cells).Full-thickness ulcers involve damage or necrosis of the epidermis,dermis, and subcutaneous tissue, and may extend into the connectivetissue below the dermis. These wounds heal by granulation (filling ofthe wound with connective tissue), contraction, and epithelialization.The most severe category of ulcer involves injury to the epidermis,dermis, subcutaneous tissue, and muscle, tendon, or bone. The woundhealing process is not complete even after the wound has closed. Theprocess of rebuilding normal skin and tissue in a wound can take up totwo years after the initial injury.

Treatment of chronic wounds varies with the severity of the wound.Partial- and full-thickness wounds are typically treated with dressingsand debridement (use of chemicals or surgery to clear away necrotic, ordead, tissue). Antibiotics may be used in the event of an infection.Partial-thickness to full-thickness wounds represent the largestcategories of chronic wound patients, the areas of greatest unmetmedical need, and the categories most amenable to treatment withprescription growth factor therapy such as Repifermin. Patients withfull-thickness wounds extending into muscle, tendon or bone are atsignificant risk of sepsis and are typically treated with surgery.

Despite the number of conservative therapies available, chronic woundsremain a very frustrating problem for health care practitioners becauseof the time-consuming nature of treatment regimens and patientnon-compliance. What is needed is a therapy that can increase apractitioner's success in healing chronic wounds and/or accelerate therate of chronic wound healing.

SUMMARY OF THE INVENTION

The present invention relates generally to compositions for woundclosure. More specifically, the present invention provides human skinequivalents engineered to express exogenous polypeptides (e.g.,antimicrobial polypeptides and keratinocyte growth factor 2) andcompositions and methods for making human skin equivalents engineered toexpress exogenous polypeptides. In addition, the present inventionprovides methods for treatment of wounds with human skin equivalentsengineered to express exogenous polypeptides.

Accordingly, in some embodiments, the present invention provides methodsfor providing cells expressing heterologous KGF-2 comprising: a)providing a host cell selected from the group consisting of primarykeratinocytes and immortalized keratinocytes and an expression vectorcomprising a DNA sequence encoding KGF-2 operably linked to a regulatorysequence; b) introducing the expression vector to the host cell (e.g.,under conditions such that said expression vector is internalized by thehost cell); and c) culturing the host cells under conditions such thatKGF-2 is expressed. The present invention is not limited to the use ofany particular primary or immortalized keratinocytes. In some preferredembodiments, the keratinocytes are NIKS cells or cell derived from NIKScells. In other embodiments, the keratinocytes are capable ofstratifying into squamous epithelia. In still other embodiments, themethods include the step of co-culturing the host cells with cellsderived from a patient. The present invention is not limited to the useof any particular expression vector. In some embodiments, the expressionvector further comprises a selectable marker. The present invention isnot limited to the use of any particular regulatory sequence. In someembodiments, the regulatory sequence is a promoter sequence. The presentinvention is not limited to any particular promoter sequence. In someembodiments, the promoter sequence is K14 promoter sequence, preferablya full-length K14 promoter sequence. In other embodiments, the promoteris an involucrin promoter. In preferred embodiments, the promotersequence allows expression in a keratinocyte. In still furtherembodiments, the present invention provides host cells produced by theforegoing method.

In some embodiments, the present invention provides compositionscomprising host cells expressing heterologous KGF-2, wherein the hostcells are selected from the group consisting of primary and immortalizedkeratinocytes. In some embodiments, the host cells are NIKS cells orcell derived from NIKS cells. In further embodiments, the KGF-2 is fulllength KGF-2.

In further embodiments, the present invention provides methods oftreating wounds comprising: a) providing immortalized keratinocytesexpressing heterologous KGF-2, and a subject with a wound; and b)contacting the wound with the immortalized cells expressing heterologousKGF-2. The present invention is not limited to any particular type ofcontacting. Indeed, a variety of ways of contacting are contemplated. Insome embodiments, the contacting comprises topical application. In otherembodiments, the contacting comprises engraftment. In still otherembodiments, the contacting comprises wound dressing. The presentinvention is not limited to the treatment of any particular type ofwound. Indeed, the treatment of a variety of wounds is contemplated,including, but not limited to those selected from the group comprisingvenous ulcers, diabetic ulcers, pressure ulcers, burns, ulcerativecolitis, mucosal injuries, internal injuries, external injuries. In someembodiments, the immortalized keratinocytes are NIKS cells. In furtherembodiments, the immortalized keratinocytes are incorporated into ahuman skin equivalent. In still further embodiments, the human skinequivalent further comprises cells derived from a patient. In otherembodiments, the methods further comprise the step mixing thekeratinocytes expressing heterologous KGF-2 with cells derived from thesubject prior to the contacting step.

In still other embodiments, the present invention provides vectorscomprising a keratinocyte specific promoter operably linked to a DNAsequence encoding KGF-2. In some embodiments, the keratinocyte specificpromoter is the K14 promoter or the involucrin promoter. The presentinvention also provides host cells and skin equivalents comprising thesevectors.

In other embodiments, the present invention provides a method forproviding a tissue (e.g., human skin equivalent) expressing an exogenousantimicrobial polypeptide or peptide comprising providing a keratinocyteand an expression vector comprising a DNA sequence encoding anantimicrobial polypeptide or peptide thereof operably linked to aregulatory sequence; introducing the expression vector into thekeratinocyte; and incorporating the keratinocyte into a tissue (e.g.,human skin equivalent). In some embodiments, the keratinocyte is capableof stratifying into squamous epithelia. In some embodiments, thekeratinocyte is selected a primary or immortalized keratinocyte (e.g.preferably NIKS cells). In certain embodiments, the expression vectorfurther comprises a selectable marker. In some preferred embodiments,the regulatory sequence is a promoter sequence (e.g., an involucrinpromoter or a keratin-14 promoter). In certain preferred embodiments,the promoter sequence allows antimicrobial polypeptide expression in thehost cell. The present invention is not limited to a particularantimicrobial polypeptide. Indeed, a variety of antimicrobialpolypeptides is contemplated including, but not limited to, human betadefensin 1, 2, and 3 and human cathelicidin. In some embodiments, thehuman beta defensin 3 has a mutated amino acid sequence (e.g., one ormore single amino acid substitutions). In some preferred embodiments,the one or more single amino acid substitutions comprise Cys40Ala,Cys45Ala, Cys55Ala, Cys62Ala, and Cys63Ala. In other embodiments, thesingle amino acid substitution is Gly38Ala. In particularly preferredembodiments, the mutated human beta defensin 3 has antimicrobialactivity. In other embodiments, the expression vector further comprisesa nucleic acid sequence encoding a signal secretion peptide. Inpreferred embodiments, the skin equivalent exhibits antimicrobialactivity. The present invention additionally provides a skin equivalentproduced by the method described herein.

In yet other embodiments, the present invention provides a compositioncomprising keratinocytes (e.g., primary or immortalized keratinocytes)expressing an exogenous antimicrobial polypeptide. In preferredembodiments, the keratinocytes are NIKS cells or cells derived from NIKScells. The present invention is not limited to a particularantimicrobial polypeptide. Indeed, a variety of antimicrobialpolypeptides is contemplated including, but not limited to, human betadefensin 1, 2, and 3 and human cathelicidin. In some embodiments, thehuman beta defensin 3 has a mutated amino acid sequence (e.g., one ormore single amino acid substitutions). In some preferred embodiments,the one or more single amino acid substitutions comprise Cys40Ala,Cys45Ala, Cys55Ala, Cys62Ala, and Cys63Ala. In other embodiments, thesingle amino acid substitution is Gly38Ala. In some embodiments, thekeratinocytes are stratified. In other embodiments, the compositionfurther comprises a dermal equivalent. In yet other embodiments, thepresent invention provides an organotypic culture of the keratinocytes.In other embodiments, the composition further comprises cells derivedfrom a patient. In still further embodiments, the composition furthercomprises keratinocytes that do not express the exogenous antimicrobialpolypeptide. In yet other embodiments, the composition further compriseskeratinocytes expressing at least one additional exogenous (e.g.,antimicrobial) polypeptide.

The present invention further provides a method of treating woundscomprising: providing primary or immortalized keratinocytes (e.g., NIKScells) expressing a exogenous antimicrobial polypeptide, and a subjectwith a wound; contacting the wound with the immortalized keratinocytesexpressing an exogenous antimicrobial polypeptide. The present inventionis not limited to a particular antimicrobial polypeptide. Indeed, avariety of antimicrobial polypeptides is contemplated including, but notlimited to, human beta defensin 1, 2, and 3 and human cathelicidin. Insome embodiments, the human beta defensin 3 has a mutated amino acidsequence (e.g., one or more single amino acid substitutions). In somepreferred embodiments, the one or more single amino acid substitutionscomprise Cys40Ala, Cys45Ala, Cys55Ala, Cys62Ala, and Cys63Ala. In otherembodiments, the single amino acid substitution is Gly38Ala. In someembodiments, the contacting comprises engraftment, topical application,or wound dressing. The present invention contemplates treatment of anytype of wound, including, but not limited to, venous ulcers, diabeticulcers, pressure ulcers, burns, ulcerative colitis, mucousal injuries,internal injuries, and external injuries. In some embodiments, the humanskin equivalent further comprises cells derived from a patient.

The present invention additionally provides a vector comprising akeratinocyte specific promoter (e.g., involucrin promoter or thekeratin-14 promoter) operably linked to a DNA sequence encoding anantimicrobial polypeptide. The present invention is not limited to aparticular antimicrobial polypeptide. Indeed, a variety of antimicrobialpolypeptides is contemplated including, but not limited to, human betadefensin 1, 2, and 3 and human cathelicidin. In some embodiments, thehuman beta defensin 3 has a mutated amino acid sequence (e.g., one ormore single amino acid substitutions). The present invention furtherprovides a host cell comprising the vector. The present invention alsoprovides a human tissue (e.g., skin equivalent) comprising the hostcell. In some embodiments, the human tissue (e.g., skin equivalent)further comprises cells derived from a patient. In other embodiments,the human tissue (e.g., skin equivalent) further comprises keratinocytesnot comprising the vector. In yet other embodiments, the human skinequivalent further comprises keratinocytes expressing at least oneadditional antimicrobial polypeptide.

In yet other embodiments, the present invention provides a method forproviding a human tissue (e.g., skin equivalent) expressing an exogenousKGF-2 and an exogenous antimicrobial polypeptide comprising providing akeratinocyte; a first expression vector comprising a DNA sequenceencoding an antimicrobial polypeptide operably linked to a regulatorysequence; and a second expression vector comprising a DNA encoding anexogenous KGF-2 polypeptide; and introducing the expression vector intothe keratinocyte; and incorporating the keratinocyte into a human tissue(e.g., skin equivalent).

In still other embodiments, the present invention provides a method ofselecting cells with increased pluripotency or multipotency relative toa population, comprising providing a population of cells;electroporating the cells under conditions such that electroporatedcells with increased pluripotency or multipotency relative to thepopulation of cells are selected. In some embodiments, theelectroporated cells exhibit stem cell like properties. In someembodiments, the population of cells are keratinocytes and theelectroporated keratinocytes have holoclone or meroclone cellmorphology. In other embodiments, the electroporated cells exhibitextended proliferative capacity. In some embodiments, the population ofcells is electroporated with an exogenous nucleic acid expressing aselectable marker. In certain embodiments, the method further comprisesthe step of culturing the cells under conditions such that only cellsexpressing the selectable marker are selected for. The present inventionadditionally provides a cell or population of cells generated by themethod.

In certain embodiments, the present invention provides a method ofselecting keratinocytes with holoclone or meroclone cell morphology,comprising providing a population of keratinocytes; and electroporatingthe keratinocytes under conditions such that electroporatedkeratinocytes with holoclone or meroclone cell morphology are selected.In some embodiments, the holoclone cell morphology comprises one or moreproperties selected from the group consisting of tightly packed cells,cells uniform in size, colonies with smooth colony edges, and an overallround colony morphology. In some embodiments, the population ofkeratinocytes is electroporated with an exogenous nucleic acidexpressing a selectable marker. In certain embodiments, the methodfurther comprises the step of culturing the keratinocytes underconditions such that only cells expressing the selectable marker areselected for. The present invention also provides a keratinocytepopulation generated by the method.

A method for providing tissues expressing heterologous KGF-2 and/orantimicrobial polypeptide comprising providing a tissue and anexpression vector comprising a DNA sequence encoding KGF-2 and/orantimicrobial polypeptide operably linked to a regulatory sequence;introducing said expression vector to said tissue under conditions suchthat said expression vector is internalized by a host cell contained insaid tissue and said KGF-2 and/or antimicrobial polypeptide isexpressed. In some embodiments, the tissue is a human tissue (e.g., ahuman skin equivalent). In some embodiments, the expression vector isintroduced to the tissue by particle bombardment, electroporation, ortransfection.

DESCRIPTION OF THE FIGURES

FIG. 1 (SEQ ID NO: 120) provides the consensus sequence of the K14promoter.

FIG. 2 provides a diagram of the construction of the K14-luciferasevector.

FIG. 3 provides a diagram of the K14-KGF-2 vector.

FIG. 4 provides a diagram of the RT-PCR strategy.

FIG. 5 provides a diagram of a vector for the expression of KGF-2 by theInvolucrin promoter.

FIG. 6 provides the DNA sequence for human beta defensin 1 (SEQ IDNO:9).

FIG. 7 provides the DNA sequence for human beta defensin 2 (SEQ IDNO:10).

FIG. 8 provides the DNA sequence for human beta defensin 3 (SEQ IDNO:11).

FIG. 9 provides the DNA sequence for the involucrin promoter (SEQ ID NO:12).

FIG. 10 (SEQ ID NOS: 121-123) provides amino acid sequence alignments ofthe human β-defensins 1-3

FIG. 11 is a schematic drawing demonstrating characteristic β-defensincovalent cysteine disulfide bond formation.

FIG. 12 is a restriction enzyme map of the human β-defensin-1 mammalianexpression vector.

FIG. 13 provides the cloning strategy for the human β-defensin vectors.

FIG. 14 describes expression vectors for expression of human β-defensin.

FIG. 15 provides the results of a RT-PCR assay for expression of humanβ-defensin mRNA.

FIG. 16 provides the results of immunoblot detection of human β-defensinprotein.

FIG. 17 shows the antimicrobial activity of human β-defensins 1, 2, and3.

FIG. 18 shows the antimicrobial activity of human β-defensins 3 in anorganotypic culture.

FIG. 19 shows a linear map and restriction digest analysis of the hCAP18vector.

FIG. 20 shows the results of a RT-PCR assay for expression of hCAP18.

DEFINITIONS

As used herein, the term “growth factor” refers to extracellularmolecules that bind to a cell-surface triggering an intracellularsignaling pathway leading to proliferation, differentiation, or othercellular response. Examples of growth factors include, but are notlimited to, growth factor I, trophic factor, Ca²⁺, insulin, hormones,synthetic molecules, pharmaceutical agents, and LDL.

As used herein, the term “keratinocyte growth factor” or “KGF” refers toa member of a group of structurally distinct proteins known as FGFs thatdisplay varying degrees of sequence homology, suggesting that they areencoded by a related family of genes. The FGFs share common receptorsites on cell surfaces. KGF, for example, can bind to FGFR-3.

As used herein, the term “antimicrobial polypeptide” refers topolypeptides and peptides thereof that inhibit the growth of microbes(e.g., bacteria). Examples of antimicrobial polypeptides include, butare not limited to, the polypeptides described in Table 1 below (e.g.,defensins or cathelicidins). Antimicrobial polypeptides include peptidessynthesized from both L-amino and D-amino acids. “Antimicrobialpolypeptides” also include peptide portions of the antimicrobialpolypeptide, obtained by any method (e.g., synthesized or enzymaticallyobtained).

As used herein, the term “defensin” refers to a family of highlycross-linked, structurally homologous antimicrobial peptides that aregenerally, but not necessarily, found in the azurophil granules ofpolymorphonuclear leukocytes (PMN's) with homologous peptides beingpresent in macrophages.

As used herein, the terms “human beta-defensin 1” or “hBD1”, when usedin reference to a protein or nucleic acid refers to a protein or nucleicacid encoding a protein that shares greater than about 50% identity withSEQ ID NO: 13 and also has at least one activity of wild type hBD1.Thus, the term hBD1 protein encompasses both proteins that are identicalto wild-type hBD1 protein and those that are derived from wild type hBD1protein (e.g., variants of hBD1 protein or chimeric genes constructedwith portions of hBD1 protein coding regions).

As used herein, the term “activity of hBD1” refers to any activity ofwild type hBD1 protein (e.g., antimicrobial activity). The term isintended to encompass all activities of hBD1 protein, alone or incombination.

In particular, the term “hBD1 gene” refers to the full-length hBD1nucleotide sequence (e.g., contained in SEQ ID NO:9). However, it isalso intended that the term encompass fragments of the hBD1 sequence, aswell as other domains within the full-length hBD1 nucleotide sequence,as well as variants of hBD1. Furthermore, the terms “hBD1 genenucleotide sequence” or “hBD1 gene polynucleotide sequence” encompassesDNA, cDNA, and RNA (e.g., mRNA) sequences.

As used herein, the terms “human beta-defensin 2” or “hBD2”, when usedin reference to a protein or nucleic acid refers to a protein or nucleicacid encoding a protein that shares greater than about 50% identity withSEQ ID NO:14 and also has at least one activity of wild type hBD2. Thus,the term hBD2 protein encompasses both proteins that are identical towild-type hBD2 protein and those that are derived from wild type hBD2protein (e.g., variants of hBD2 protein or chimeric genes constructedwith portions of hBD2 protein coding regions).

As used herein, the term “activity of hBD2” refers to any activity ofwild type hBD2 protein (e.g., antimicrobial activity). The term isintended to encompass all activities of hBD2 protein, alone or incombination.

In particular, the term “hBD2 gene” refers to the full-length hBD1nucleotide sequence (e.g., contained in SEQ ID NO:10). However, it isalso intended that the term encompass fragments of the hBD1 sequence, aswell as other domains within the full-length hBD2 nucleotide sequence,as well as variants of hBD1. Furthermore, the terms “hBD2 genenucleotide sequence” or “hBD1 gene polynucleotide sequence” encompassesDNA, cDNA, and RNA (e.g., mRNA) sequences.

As used herein, the terms “human beta-defensin 3” or “hBD3”, when usedin reference to a protein or nucleic acid refers to a protein or nucleicacid encoding a protein that shares greater than about 50% identity withSEQ ID NO:15 and also has at least one activity of wild type hBD3. Thus,the term hBD3 protein encompasses both proteins that are identical towild-type hBD3 protein and those that are derived from wild type hBD3protein (e.g., variants of hBD3 protein or chimeric genes constructedwith portions of hBD3 protein coding regions).

As used herein, the term “activity of hBD3” refers to any activity ofwild type hBD3 protein (e.g., antimicrobial activity). The term isintended to encompass all activities of hBD1 protein, alone or incombination.

In particular, the term “hBD3 gene” refers to the full-length hBD3nucleotide sequence (e.g., contained in SEQ ID NO:11). However, it isalso intended that the term encompass fragments of the hBD3 sequence, aswell as other domains within the full-length hBD3 nucleotide sequence,as well as variants of hBD3. Furthermore, the terms “hBD3 genenucleotide sequence” or “hBD3 gene polynucleotide sequence” encompassesDNA, cDNA, and RNA (e.g., mRNA) sequences.

As used herein, the term “NIKS cells” refers to cells having thecharacteristics of the cells deposited as cell line ATCC CRL-12191.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., GKLF). The polypeptide can be encoded by a fulllength coding sequence or by any portion of the coding sequence so longas the desired activity or functional properties (e.g., enzymaticactivity, ligand binding, signal transduction, etc.) of the full-lengthor fragment are retained. The term also encompasses the coding region ofa structural gene and the including sequences located adjacent to thecoding region on both the 5′ and 3′ ends for a distance of about 1 kb oneither end such that the gene corresponds to the length of thefull-length mRNA. The sequences that are located 5′ of the coding regionand which are present on the mRNA are referred to as 5′ untranslatedsequences. The sequences that are located 3′ or downstream of the codingregion and that are present on the mRNA are referred to as 3′untranslated sequences. The term “gene” encompasses both cDNA andgenomic forms of a gene. A genomic form or clone of a gene contains thecoding region interrupted with non-coding sequences termed “introns” or“intervening regions” or “intervening sequences.” Introns are segmentsof a gene that are transcribed into nuclear RNA (hnRNA); introns maycontain regulatory elements such as enhancers. Introns are removed or“spliced out” from the nuclear or primary transcript; introns thereforeare absent in the messenger RNA (mRNA) transcript. The mRNA functionsduring translation to specify the sequence or order of amino acids in anascent polypeptide.

As used herein, the terms “nucleic acid molecule encoding,” “DNAsequence encoding,” and “DNA encoding” refer to the order or sequence ofdeoxyribonucleotides along a strand of deoxyribonucleic acid. The orderof these deoxyribonucleotides determines the order of amino acids alongthe polypeptide (protein) chain. The DNA sequence thus codes for theamino acid sequence.

As used herein, the term “recombinant DNA molecule” as used hereinrefers to a DNA molecule that is comprised of segments of DNA joinedtogether by means of molecular biological techniques.

The term “isolated” when used in relation to a nucleic acid, as in “anisolated oligonucleotide” or “isolated polynucleotide” refers to anucleic acid sequence that is identified and separated from at least onecontaminant nucleic acid with which it is ordinarily associated in itsnatural source. Isolated nucleic acid is present in a form or settingthat is different from that in which it is found in nature. In contrast,non-isolated nucleic acids are nucleic acids such as DNA and RNA foundin the state they exist in nature. For example, a given DNA sequence(e.g., a gene) is found on the host cell chromosome in proximity toneighboring genes; RNA sequences, such as a specific mRNA sequenceencoding a specific protein, are found in the cell as a mixture withnumerous other mRNAs that encode a multitude of proteins. However,isolated nucleic acid encoding KGF-2 includes, by way of example, suchnucleic acid in cells ordinarily expressing KGF-2 where the nucleic acidis in a chromosomal location different from that of natural cells, or isotherwise flanked by a different nucleic acid sequence than that foundin nature. The isolated nucleic acid, oligonucleotide, or polynucleotidemay be present in single-stranded or double-stranded form. When anisolated nucleic acid, oligonucleotide or polynucleotide is to beutilized to express a protein, the oligonucleotide or polynucleotidewill contain at a minimum the sense or coding strand (i.e., theoligonucleotide or polynucleotide may be single-stranded), but maycontain both the sense and anti-sense strands (i.e., the oligonucleotideor polynucleotide may be double-stranded).

As used herein the term “portion” when in reference to a nucleotidesequence (as in “a portion of a given nucleotide sequence”) refers tofragments of that sequence. The fragments may range in size from fournucleotides to the entire nucleotide sequence minus one nucleotide (10nucleotides, 20, 30, 40, 50, 100, 200, etc.).

As used herein the term “coding region” when used in reference tostructural gene refers to the nucleotide sequences that encode the aminoacids found in the nascent polypeptide as a result of translation of amRNA molecule. The coding region is bounded, in eukaryotes, on the 5′side by the nucleotide triplet “ATG” that encodes the initiatormethionine and on the 3′ side by one of the three triplets that specifystop codons (i.e., TAA, TAG, TGA).

As used herein, the term “purified” or “to purify” refers to the removalof contaminants from a sample.

As used herein, the term “vector” is used in reference to nucleic acidmolecules that transfer DNA segment(s) from one cell to another. Theterm “vehicle” is sometimes used interchangeably with “vector.”

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

A “regulatory sequence” refers to a polynucleotide sequence that isnecessary for regulation of expression of a coding sequence to which thepolynucleotide sequence is operably linked. The nature of suchregulatory sequences differs depending upon the host organism. Inprokaryotes, such regulatory sequences generally include, for example, apromoter, and/or a transcription termination sequence. In eukaryotes,generally, such regulatory sequences include, for example, a promoterand/or a transcription termination sequence. The term “regulatorysequence” may also include additional components the presence of whichare advantageous, for example, a secretory leader sequence for secretionof the polypeptide attached thereto.

“Operably linked” refers to a juxtaposition wherein the components sodescribed are in a relationship permitting them to function in theirintended manner. A regulatory sequence is “operably linked” to a codingsequence when it is joined in such a way that expression of the codingsequence is achieved under conditions compatible with the regulatorysequence.

“PCR” refers to the techniques of the polymerase chain reaction asdescribed in Saiki, et al., Nature 324:163 (1986); and Scharf et al.,Science 233:1076-1078 (1986); U.S. Pat. No. 4,683,195; and U.S. Pat. No.4,683,202. As used herein, x is “heterologous” with respect to y if x isnot naturally associated with y or x is not associated with y in thesame manner as is found in nature.

By “pharmaceutically acceptable carrier,” is meant any carrier that isused by persons in the art for administration into a human that does notitself induce any undesirable side effects such as the production ofantibodies, fever, etc. Suitable carriers are typically large, slowlymetabolized macromolecules that can be a protein, a polysaccharide, apolylactic acid, a polyglycolic acid, a polymeric amino acid, amino acidcopolymers or an inactive virus particle. Such carriers are well knownto those of ordinary skill in the art. Preferably the carrier isthyroglobulin.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

The terms “overexpression” and “overexpressing” and grammaticalequivalents, are used in reference to levels of mRNA to indicate a levelof expression approximately 3-fold higher than that typically observedin a given tissue in a control or non-transgenic animal. Levels of mRNAare measured using any of a number of techniques known to those skilledin the art including, but not limited to Northern blot analysis.Appropriate controls are included on the Northern blot to control fordifferences in the amount of RNA loaded from each tissue analyzed (e.g.,the amount of 28S rRNA, an abundant RNA transcript present atessentially the same amount in all tissues, present in each sample canbe used as a means of normalizing or standardizing the KGF-2mRNA-specific signal observed on Northern blots). The amount of mRNApresent in the band corresponding in size to the correctly spliced KGF-2transgene RNA is quantified; other minor species of RNA which hybridizeto the transgene probe are not considered in the quantification of theexpression of the transgenic mRNA.

The term “transfection” as used herein refers to the introduction offoreign DNA into eukaryotic cells. Transfection may be accomplished by avariety of means known to the art including calcium phosphate-DNAco-precipitation, DEAE-dextran-mediated transfection, polybrene-mediatedtransfection, electroporation, microinjection, liposome fusion,lipofection, protoplast fusion, retroviral infection, and biolistics.

The term “stable transfection” or “stably transfected” refers to theintroduction and integration of foreign DNA into the genome of thetransfected cell. The term “stable transfectant” refers to a cell thathas stably integrated foreign DNA into the genomic DNA.

The term “transient transfection” or “transiently transfected” refers tothe introduction of foreign DNA into a cell where the foreign DNA doesnot integrate into the genome of the transfected cell. The foreign DNApersists in the nucleus of the transfected cell for several days. Duringthis time the foreign DNA is subject to the regulatory controls thatgovern the expression of endogenous genes in the chromosomes. The term“transient transfectant” refers to cells that have taken up foreign DNAbut have failed to integrate this DNA.

The term “calcium phosphate co-precipitation” refers to a technique forthe introduction of nucleic acids into a cell. The uptake of nucleicacids by cells is enhanced when the nucleic acid is presented as acalcium phosphate-nucleic acid co-precipitate. The original technique ofGraham and van der Eb (Graham and van der Eb, Virol., 52:456 [1973]) hasbeen modified by several groups to optimize conditions for particulartypes of cells. The art is well aware of these numerous modifications.

The term “test compound” refers to any chemical entity, pharmaceutical,drug, and the like that can be used to treat or prevent a disease,illness, sickness, or disorder of bodily function, or otherwise alterthe physiological or cellular status of a sample. Test compoundscomprise both known and potential therapeutic compounds. A test compoundcan be determined to be therapeutic by screening using the screeningmethods of the present invention. A “known therapeutic compound” refersto a therapeutic compound that has been shown (e.g., through animaltrials or prior experience with administration to humans) to beeffective in such treatment or prevention.

The term “sample” as used herein is used in its broadest sense. A samplesuspected of containing a human chromosome or sequences associated witha human chromosome may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern blot analysis), RNA (insolution or bound to a solid support such as for Northern blotanalysis), cDNA (in solution or bound to a solid support) and the like.A sample suspected of containing a protein may comprise a cell, aportion of a tissue, an extract containing one or more proteins and thelike.

As used herein, the term “response”, when used in reference to an assay,refers to the generation of a detectable signal (e.g., accumulation ofreporter protein, increase in ion concentration, accumulation of adetectable chemical product).

As used herein, the term “reporter gene” refers to a gene encoding aprotein that may be assayed. Examples of reporter genes include, but arenot limited to, luciferase (See, e.g., deWet et al., Mol. Cell. Biol.7:725 [1987] and U.S. Pat. Nos. 6,074,859; 5,976,796; 5,674,713; and5,618,682; all of which are incorporated herein by reference), greenfluorescent protein (e.g., GenBank Accession Number U43284; a number ofGFP variants are commercially available from CLONTECH Laboratories, PaloAlto, Calif.), chloramphenicol acetyltransferase, Beta-galactosidase,alkaline phosphatase, and horse radish peroxidase.

DETAILED DESCRIPTION

The present invention provides human skin equivalents (e.g., NIKS cells)expressing exogenous polypeptides (e.g., KGF-2 and antimicrobialpolypeptides), and compositions and methods for making such cells. Inaddition, the present invention provides methods for treatment of woundswith such cells.

I. Methods of Generating Host Cells

In some embodiments, the present invention provides methods ofgenerating human tissues such as skin equivalents (e.g., from NIKScells) expressing exogenous polypeptides (e.g., KGF-2 and antimicrobialpolypeptides).

A) Host Cells

Generally, any source of cells or cell line that can stratify intosquamous epithelia is useful in the present invention. Accordingly, thepresent invention is not limited to the use of any particular source ofcells that are capable of differentiating into squamous epithelia.Indeed, the present invention contemplates the use of a variety of celllines and sources that can differentiate into squamous epithelia,including both primary and immortalized keratinocytes. Sources of cellsinclude keratinocytes and dermal fibroblasts biopsied from humans andcavaderic donors (Auger et al., In Vitro Cell. Dev. Biol.—Animal36:96-103; U.S. Pat. Nos. 5,968,546 and 5,693,332, each of which isincorporated herein by reference), neonatal foreskins (Asbill et al.,Pharm. Research 17(9): 1092-97 (2000); Meana et al., Burns 24:621-30(1998); U.S. Pat. Nos. 4,485,096; 6,039,760; and 5,536,656, each ofwhich is incorporated herein by reference), and immortalizedkeratinocytes cell lines such as NM1 cells (Baden, In Vitro Cell. Dev.Biol. 23(3):205-213 (1987)), HaCaT cells (Boucamp et al., J. cell. Biol.106:761-771 (1988)); and NIKS cells (Cell line BC-1-Ep/SL; U.S. Pat. No.5,989,837, incorporated herein by reference; ATCC CRL-12191). Each ofthese cell lines can be cultured or genetically modified as describedbelow in order to produce a cell line capable of expressing an exogenouspolypeptide.

In particularly preferred embodiments, NIKS cells or cells derived fromNIKS cells are utilized. NIKS cells (Cell line BC-1-Ep/SL; U.S. Pat.Nos. 5,989,837, 6,514,711, 6,495,135, 6,485,724, and 6,214,567; each ofwhich is incorporated herein by reference; ATCC CRL-12191). Thediscovery of a novel human keratinocyte cell line (near-diploidimmortalized keratinocytes or NIKS) provides an opportunity togenetically engineer human keratinocytes for new therapeutic methods. Aunique advantage of the NIKS cells is that they are a consistent sourceof genetically-uniform, pathogen-free human keratinocytes. For thisreason, they are useful for the application of genetic engineering andgenomic gene expression approaches to provide skin equivalent cultureswith properties more similar to human skin. Such systems will provide animportant alternative to the use of animals for testing compounds andformulations. The NIKS keratinocyte cell line, identified andcharacterized at the University of Wisconsin, is nontumorigenic,exhibits a stable karyotype, and undergoes normal differentiation bothin monolayer and organotypic culture. NIKS cells form fully stratifiedskin equivalents in culture. These cultures are indistinguishable by allcriteria tested thus far from organotypic cultures formed from primaryhuman keratinocytes. Unlike primary cells however, the immortalized NIKScells will continue to proliferate in monolayer culture indefinitely.This provides an opportunity to genetically manipulate the cells andisolate new clones of cells with new useful properties (Allen-Hoffmannet al., J. Invest. Dermatol., 114(3): 444-455 (2000)).

The NIKS cells arose from the BC-1-Ep strain of human neonatal foreskinkeratinocytes isolated from an apparently normal male infant. In earlypassages, the BC-1-Ep cells exhibited no morphological or growthcharacteristics that were atypical for cultured normal humankeratinocytes. Cultivated BC-1-Ep cells exhibited stratification as wellas features of programmed cell death. To determine replicative lifespan,the BC-1-Ep cells were serially cultivated to senescence in standardkeratinocyte growth medium at a density of 3×10⁵ cells per 100-mm dishand passaged at weekly intervals (approximately a 1:25 split). Bypassage 15, most keratinocytes in the population appeared senescent asjudged by the presence of numerous abortive colonies that exhibitedlarge, flat cells. However, at passage 16, keratinocytes exhibiting asmall cell size were evident. By passage 17, only the small-sizedkeratinocytes were present in the culture and no large, senescentkeratinocytes were evident. The resulting population of smallkeratinocytes that survived this putative crisis period appearedmorphologically uniform and produced colonies of keratinocytesexhibiting typical keratinocyte characteristics including cell-celladhesion and apparent squame production. The keratinocytes that survivedsenescence were serially cultivated at a density of 3×10⁵ cells per100-mm dish. Typically the cultures reached a cell density ofapproximately 8×10⁶ cells within 7 days. This stable rate of cell growthwas maintained through at least 59 passages, demonstrating that thecells had achieved immortality. The keratinocytes that emerged from theoriginal senescencing population were originally designatedBC-1-Ep/Spontaneous Line and are now termed NIKS. The NIKS cell line hasbeen screened for the presence of proviral DNA sequences for HIV-1,HIV-2, EBV, CMV, HTLV-1, HTLV-2, HBV, HCV, B-19 parvovirus, HPV-16 andHPV-31 using either PCR or Southern analysis. None of these viruses weredetected.

Chromosomal analysis was performed on the parental BC-1-Ep cells atpassage 3 and NIKS cells at passages 31 and 54. The parental BC-1-Epcells have a normal chromosomal complement of 46, XY. At passage 31, allNIKS cells contained 47 chromosomes with an extra isochromosome of thelong arm of chromosome 8. No other gross chromosomal abnormalities ormarker chromosomes were detected. At passage 54, all cells contained theisochromosome 8.

The DNA fingerprints for the NIKS cell line and the BC-1-Epkeratinocytes are identical at all twelve loci analyzed demonstratingthat the NIKS cells arose from the parental BC-1-Ep population. The oddsof the NIKS cell line having the parental BC-1-Ep DNA fingerprint byrandom chance is 4×10⁻¹⁶. The DNA fingerprints from three differentsources of human keratinocytes, ED-1-Ep, SCC4 and SCC13y are differentfrom the BC-1-Ep pattern. This data also shows that keratinocytesisolated from other humans, ED-1-Ep, SCC4, and SCC13y, are unrelated tothe BC-1-Ep cells or each other. The NIKS DNA fingerprint data providesan unequivocal way to identify the NIKS cell line.

Loss of p53 function is associated with an enhanced proliferativepotential and increased frequency of immortality in cultured cells. Thesequence of p53 in the NIKS cells is identical to published p53sequences (GenBank accession number: M14695). In humans, p53 exists intwo predominant polymorphic forms distinguished by the amino acid atcodon 72. Both alleles of p53 in the NIKS cells are wild-type and havethe sequence CGC at codon 72, which codes for an arginine. The othercommon form of p53 has a proline at this position. The entire sequenceof p53 in the NIKS cells is identical to the BC-1-Ep progenitor cells.Rb was also found to be wild-type in NIKS cells.

Anchorage-independent growth is highly correlated to tumorigenicity invivo. For this reason, the anchorage-independent growth characteristicsof NIKS cells in agar or methylcellulose-containing medium wasinvestigated. After 4 weeks in either agar- ormethylcellulose-containing medium, NIKS cells remained as single cells.The assays were continued for a total of 8 weeks to detect slow growingvariants of the NIKS cells. None were observed.

To determine the tumorigenicity of the parental BC-1-Ep keratinocytesand the immortal NIKS keratinocyte cell line, cells were injected intothe flanks of athymic nude mice. The human squamous cell carcinoma cellline, SCC4, was used as a positive control for tumor production in theseanimals. The injection of samples was designed such that animalsreceived SCC4 cells in one flank and either the parental BC-1-Epkeratinocytes or the NIKS cells in the opposite flank. This injectionstrategy eliminated animal to animal variation in tumor production andconfirmed that the mice would support vigorous growth of tumorigeniccells. Neither the parental BC-1-Ep keratinocytes (passage 6) nor theNIKS keratinocytes (passage 35) produced tumors in athymic nude mice.

NIKS cells were analyzed for the ability to undergo differentiation inboth surface culture and organotypic culture. For cells in surfaceculture, a marker of squamous differentiation, the formation cornifiedenvelopes was monitored. In cultured human keratinocytes, early stagesof cornified envelope assembly result in the formation of an immaturestructure composed of involucrin, cystatin-α and other proteins, whichrepresent the innermost third of the mature cornified envelope. Lessthan 2% of the keratinocytes from the adherent BC-1-Ep cells or the NIKScell line produce cornified envelopes. This finding is consistent withprevious studies demonstrating that actively growing, subconfluentkeratinocytes produce less than 5% cornified envelopes. To determinewhether the NIKS cell line is capable of producing cornified envelopeswhen induced to differentiate, the cells were removed from surfaceculture and suspended for 24 hours in medium made semi-solid withmethylcellulose. Many aspects of terminal differentiation, includingdifferential expression of keratins and cornified envelope formation canbe triggered in vitro by loss of keratinocyte cell-cell andcell-substratum adhesion. The NIKS keratinocytes produced as many as andusually more cornified envelopes than the parental keratinocytes. Thesefindings demonstrate that the NIKS keratinocytes are not defective intheir ability to initiate the formation of this cell type-specificdifferentiation structure.

To confirm that the NIKS keratinocytes can undergo squamousdifferentiation, the cells were cultivated in organotypic culture.Keratinocyte cultures grown on plastic substrata and submerged in mediumreplicate but exhibit limited differentiation. Specifically, humankeratinocytes become confluent and undergo limited stratificationproducing a sheet consisting of 3 or more layers of keratinocytes. Bylight and electron microscopy there are striking differences between thearchitecture of the multilayered sheets formed in tissue culture andintact human skin. In contrast, organotypic culturing techniques allowfor keratinocyte growth and differentiation under in vivo-likeconditions. Specifically, the cells adhere to a physiological substratumconsisting of dermal fibroblasts embedded within a fibrillar collagenbase. The organotypic culture is maintained at the air-medium interface.In this way, cells in the upper sheets are air-exposed while theproliferating basal cells remain closest to the gradient of nutrientsprovided by diffusion through the collagen gel. Under these conditions,correct tissue architecture is formed. Several characteristics of anormal differentiating epidermis are evident. In both the parental cellsand the NIKS cell line a single layer of cuboidal basal cells rests atthe junction of the epidermis and the dermal equivalent. The roundedmorphology and high nuclear to cytoplasmic ratio is indicative of anactively dividing population of keratinocytes. In normal humanepidermis, as the basal cells divide they give rise to daughter cellsthat migrate upwards into the differentiating layers of the tissue. Thedaughter cells increase in size and become flattened and squamous.Eventually these cells enucleate and form cornified, keratinizedstructures. This normal differentiation process is evident in the upperlayers of both the parental cells and the NIKS cells. The appearance offlattened squamous cells is evident in the upper layers of keratinocytesand demonstrates that stratification has occurred in the organotypiccultures. In the uppermost part of the organotypic cultures theenucleated squames peel off the top of the culture. To date, nohistological differences in differentiation at the light microscopelevel between the parental keratinocytes and the NIKS keratinocyte cellline grown in organotypic culture have been observed.

To observe more detailed characteristics of the parental (passage 5) andNIKS (passage 38) organotypic cultures and to confirm the histologicalobservations, samples were analyzed using electron microscopy. Parentalcells and the immortalized human keratinocyte cell line, NIKS, wereharvested after 15 days in organotypic culture and sectionedperpendicular to the basal layer to show the extent of stratification.Both the parental cells and the NIKS cell line undergo extensivestratification in organotypic culture and form structures that arecharacteristic of normal human epidermis. Abundant desmosomes are formedin organotypic cultures of parental cells and the NIKS cell line. Theformation of a basal lamina and associated hemidesmosomes in the basalkeratinocyte layers of both the parental cells and the cell line wasalso noted.

Hemidesmosomes are specialized structures that increase adhesion of thekeratinocytes to the basal lamina and help maintain the integrity andstrength of the tissue. The presence of these structures was especiallyevident in areas where the parental cells or the NIKS cells had attacheddirectly to the porous support. These findings are consistent withearlier ultrastructural findings using human foreskin keratinocytescultured on a fibroblast-containing porous support. Analysis at both thelight and electron microscopic levels demonstrate that the NIKS cellline in organotypic culture can stratify, differentiate, and formstructures such as desmosomes, basal lamina, and hemidesmosomes found innormal human epidermis.

B) KGF-2

In some embodiments, the present invention provides human skinequivalents (e.g., keratinocytes) that express exogenous KGF-2 protein.KGF-2 is a 208 amino acid protein that influences normal keratinocyteand epithelial cells to proliferate and migrate to wound sites. Proteinand nucleic acid sequences for KGF-2 are provided in U.S. Pat. No.6,077,692; which is incorporated herein by reference.

KGF-2 promotes wound healing in tissues containing keratinocytes andfibroblasts by having a positive proliferative effect on epithelialcells and mediating keratinocyte migration. In addition, KGF-2 promoteswound healing by increasing deposition of granulation tissue andcollagen, and maturation of collagen (Soler et al., Wound Repair Regen.7(3):172-178 (1999)).

C) Antimicrobial Polypeptides

In some embodiments, the present invention provides human skinequivalents (e.g., keratinocytes) that express exogenous antimicrobialpolypeptides. In intact human skin, the stratum corneum serves as thefirst line of defense against microbial organisms. The stratum corneumis the uppermost, nonviable, desiccated layer of the epidermis that iscomposed of fully differentiated keratinocytes. The innate immuneresponse prevents invasion of microbial organisms if the outer mostlayer of the skin barrier is penetrated. This response includesphagocytosis by macrophages and neutrophils and their production ofreactive oxygen intermediates that kill microbial agents. Associatedwith this line of defense are antimicrobial peptides that are naturallyexpressed and localized to the upper layers of the epidermis. The mostthoroughly studied human antimicrobial peptides belong to twosubfamilies, the α- and β-defensins, which differ from one another bytheir disulfide bond pairing, genomic organization and tissuedistributions (Ganz, T. and J. Weiss, Semin Hematol, 1997. 34(4): p.343-54). The β-defensins are characteristically found in epithelialtissues and are expressed in human keratinocytes. This defensinsubfamily demonstrates strong antimicrobial activity against a broadspectrum of pathogenic agents, including bacteria, fungi and viruses.

Microorganisms have difficulty acquiring resistance to the defensinpeptides, making these peptides very attractive for therapeutic use asantibiotics (Schroder, J. M., Biochem Pharmacol, 1999. 57(2): p.121-34). In clinical trials, defensin peptides applied to skin have beenfound to be safe (Hancock, R. E., Lancet, 1997. 349(9049): p. 418-22).The safety of topically-applied defensins is consistent with the findingthat human epidermal keratinocytes express defensin peptides in vivo.

In the human genome, all known defensin genes cluster to a <1 Mb regionof chromosome 8p22-p23; these findings suggest an evolutionaryconservation of this gene family. Harder, J., et al., Mapping of thegene encoding human beta-defensin-2 (DEFB2) to chromosome region8p22-p23.1. Genomics, 1997. 46(3): p. 472-5. It is generally acceptedthat evolutionarily conserved genes maintain some overlap in genefunction. The defensin gene family is no exception to this theory. Thedefensin genes encode small (3-5 kDa), cationic molecules characterizedby an amphipathic structure and have six cysteine residues that formthree intramolecular disulfide bonds (see FIG. 11). These cationicregions are thought to be attractive to the anionic surfaces of mostbacteria. The human defensin gene family is divided into twosubfamilies: the α-defensins and β-defensins that differ from oneanother by their disulfide bond pairing, genomic organization and tissuedistributions. The α- and β-defensins share similarity in tertiarystructure and both contain triple stranded antiparallel beta sheets(Pardi, A., et al., Biochemistry, 1992. 31(46): p. 11357-64; Zimmermann,G. R., et al., Biochemistry, 1995. 34(41): p. 13663-71). However, theirantimicrobial mechanisms of action are distinct from one another.

Historically the α-defensins have been found in storage granules ofspecialized cell types such as neutrophils and Paneth cells of the smallintestine, whereas the β-defensins are expressed in epithelial tissues.The α-defensins also have an inhibitory pro-region in theiramino-terminal sequence, which is cleaved off after release fromgranules. The pro-region is likely to contain a granule targeting motifbut may function independently as a protease inhibitor. The broadspectrum of antimicrobial activity is mediated in part bypermeabilization of biological membranes. Although extremely potent forkilling invading microorganisms, α-defensins have also been shown to betoxic to eukaryotic cell types (Lichtenstein, A., et al., Blood, 1986.68(6): p. 1407-10; Okrent et al., Am Rev Respir Dis, 1990. 141(1): p.179-85). The α-defensin-induced pleiotropic cell killing activity makesthis subfamily of defensins unattractive as a gene candidate forexpression in living human skin substitutes.

Keratinocytes of the skin and other epithelia harbor endogenouslyexpressed members of the β-defensins. To date, there have been sixdistinct genes identified. Three of these human β-defensin genes, hBD-1,hBD-2 & hBD-3, are expressed in epidermal keratinocytes of the skin. Thefirst exon encodes the signal sequence and propeptide and the secondexon encodes the mature peptide. Amino acid sequence alignmenthighlighting conserved residues and the characteristic six cysteineresidues of the human β-defensins 1-3 are shown in FIG. 10. Thedisulfide covalent bonds required for secondary structure of the activepeptide are demonstrated in FIG. 11.

Several factors are thought to contribute to the antimicrobial action ofthe β-defensins on microbes. First because of their cationic andamphiphilic characteristics, antimicrobial peptides bind and insert intothe cytoplasmic membrane, where they assemble into multimeric pores, anddestroy the target microbe by changing membrane conductance and alteringintracellular function (White, S. H., W. C. Wimley, and M. E. Selsted,Curr Opin Struct Biol, 1995. 5(4): p. 521-7; Boman, H. G., Annu RevImmunol, 1995. 13: p. 61-92). Most antimicrobial peptides killmicroorganisms by forming pores in the cell membrane. These peptides arenot toxic to mammalian cells due to the sensitivity of these peptideantibiotics to cholesterol and phospholipids, major components ofmammalian cell membranes. The β-defensins are attractive candidates fortherapeutic use as antibiotics since it is difficult for microorganismsto acquire resistance to the peptides' bactericidal mechanism of action(Schroder, J. M., Biochem Pharmacol, 1999. 57(2): p. 121-34).

When expressed, the β-defensin peptides appear to initially localize tothe cytoplasm of undifferentiated or less differentiated keratinocytes.As these cells differentiate and move closer to the epidermal surface,they secrete these antimicrobial peptides onto the keratinocyte membraneor into the intracellular space. The signal peptide sequence is thoughtto contribute to the specialized localization of this active peptide.Finally human β-defensin peptides accumulate in the dehydrated cells ofthe epidermal surface. Studies demonstrate that, although the threeβ-defensin genes are very similar, their expression is determined bycompletely different regulatory mechanisms (Frye, M., J. Bargon, and R.Gropp, J Mol Med, 2001. 79(5-6): p. 275-82).

The burn wound is an ideal environment for bacterial growth and providesa pathway for microbial invasion. Luterman and coworkers concluded“Burned skin is a nidus and portal for bacterial invasion, causing burnwound sepsis, the leading cause of death in burn units around the world”(Luterman, A., C. C. Dacso, and P. W. Curreri, Am J Med, 1986. 81(1A):p. 45-52). Infection is further promoted by skin loss and post burnimmuno-suppression. As expected, human defensin gene expression isdiminished in full thickness burn wounds most probably due to thedestruction of the epithelium. For example, human β-defensin gene(hBD-2) expression is virtually undetectable in the burn woundsuggesting the loss of defensins due to thermal destruction of the skin(Milner, S. M. and M. R. Ortega, Burns, 1999. 25(5): p. 411-3). Aroutinely used debridement procedure may also contribute to significantremoval of epithelia in a wound bed. Debridement speeds the healing ofulcers, burns, and other wounds by removing dead tissue so that theremaining living tissue can adequately heal. Wounds that containnon-living (necrotic) tissue take longer to heal because necrotic debrisis a nutrient source for bacteria in a wound. The debridement procedureintroduces a potential risk that surface bacteria may be introduceddeeper into the body, causing infection.

Bacteria typically encountered in a burn wound include E. coli, P.aeruginosa, S. aureus, and C. albicans (Heggers, J. P., Treatment ofinfection in burns, H. DN, Editor. 1996, WB Saunders: London. p.98-135). All of these microbes are killed by one or more of theβ-defensin antimicrobial peptides.

Some β-defensin family members are upregulated in response toinflammatory stimuli or bacterial invasion. Others remainnon-responsive, downregulated or suppressed in response to inflammatorystimuli or bacterial exposure. In unwounded, intact skin, the calculatedepidermal concentrations of β-defensin peptides are well within therange needed for their antimicrobial effects. The β-defensins possesschemotactic activity for immature dendritic cells and memory T cells.These chemotactic responses require much lower concentrations thanrequired for antimicrobial activity (Yang, D., et al., Science, 1999.286(5439): p. 525-8). As a result of this cross-talk, the β-defensinsare thought to mediate an important link between innate and adaptiveimmunity. Therefore, the β-defensins appear to play a multifunctionalrole by promoting both an adaptive immune response and inflammation,while facilitating wound healing through their antimicrobial activity.Adaptive immunity is promoted through the endogenous antimicrobialpeptides in healthy human skin and likely provides an effective shieldfrom microbial infection; however, patients with unhealthy or chronicskin wounds would also benefit from boosted local antimicrobial peptidelevels.

The hBD-1 gene encodes for a 3.9 kDa basic peptide that was originallyidentified in hemofiltrates from human patients with end stage renaldisease (Bensch, K. W., et al., FEBS Lett, 1995. 368(2): p. 331-5).hBD-1 bactericidal activity is predominantly against gram negativebacteria such as E. coli and P. aeruginosa. Constitutive hBD-1expression has been observed in skin from various sites on the body. Theoverexpression of hBD-1 in immortalized human skin cells (HaCat) isassociated with keratinocyte cell differentiation. Overexpression wasconfirmed to have no effect on proliferating cells. The presentinvention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that β-defensin geneexpression is a consequence of differentiation, rather than an inducerof differentiation in keratinocytes (Frye, M., J. Bargon, and R. Gropp,J Mol Med, 2001. 79(5-6): p. 275-82). hBD-1 expression in differentiatedkeratinocyte cells is inhibited upon exposure to bacteria. The presentinvention is not limited to a particular mechanism. Indeed, anunderstanding of the mechanism is not necessary to practice the presentinvention. Nonetheless, it is contemplated that this result indicatesthat this factor is an important mediator of the healing process inregenerating epithelia. These studies confirm the upregulation of hBD-1expression is a result of factors not associated with an inflammatoryresponse. This antimicrobial peptide is not induced by inflammatorycytokines, which is consistent with the lack of cytokine-responsivetranscription factor regulatory elements in the hBD-1 5′ regulatorysequences.

hBD-2 peptide was originally identified in desquamated squames ofpsoriatic skin and hBD-2 gene expression has since been identified innormal human keratinocytes (Harder, J., et al., Genomics, 1997. 46(3):p. 472-5). This gene encodes for a 4 kDa basic peptide. Variableendogenous levels of expression have been observed when comparing skinfrom various sites on the body, with the most prominent expressionobserved in facial skin and foreskin. Expression is localized to thesuprabasal layers and the stratum corneum of intact skin. Low levels ofhBD-2 protein have been detected in the cytoplasm of keratinocytes inbasal layers of skin tissue. These proteins are believed to be secretedinto the cell membrane or intercellular spaces as the cells achieve asuprabasal position in the tissue and eventually concentrate in thedehydrated cells of the stratum corneum. hBD-2 peptide efficientlycombats clinical isolates of gram negative bacteria such as P.aeruginosa and E. coli, while only having a bacteriostatic effect, athigh concentrations, on gram positive bacterial strains such as S.aureus (Liu, A. Y., et al., J Invest Dermatol, 2002. 118(2): p. 275-81).Studies show that endogenous expression is triggered by inflammatorycytokines as well as exposure to bacteria. Finally, not only does hBD-2have antimicrobial activity, it also modulates the inflammatory responsein various skin conditions (Garcia, J. R., et al., Cell Tissue Res,2001. 306(2): p. 257-64).

The hBD-3 gene encodes for a 5 kDa basic peptide that was identified byscreening genomic sequences for antimicrobial activity and the abilityto activate monocytes. The gene was cloned from differentiatedrespiratory epithelial cells. Strongest expression has been exhibited inthe skin and tonsil. Endogenous expression is triggered by inflammation,and therefore, hBD-3 is not constitutive but rather a readily inducibleantimicrobial peptide. This peptide is also a potent chemoattractant formonocytes and neutrophils, which are strongly involved in the innateimmune response (Garcia, J. R., et al., Cell Tissue Res, 2001. 306(2):p. 257-64). hBD-3 possesses a broad spectrum antimicrobial peptideactivity at low micromolar concentrations, against many potentialpathogenic microbes including P. aeruginosa, S. pyrogenes,multiresistant S. aureus, vancomycin-resistant E. faecium, and the yeastC. albicans. hBD-3 gene expression is also induced in HaCat and culturedskin-derived keratinocytes when stimulated with heat-inactivatedbacteria (Harder, J., et al., Nature, 1997. 387(6636): p. 861). It isspeculated that some disorders of defective innate immunity, such asunexplained recurrent infections of particular organs, may be caused byabnormalities that reduce expression of one or more genes that encodedefensins or other antimicrobial peptides. Synthetic hBD-3 proteinexhibits a strong antimicrobial activity against gram-negative andgram-positive bacteria and fungi.

The present invention contemplates that the overexpression of exogenousantimicrobial polypeptides in human skin equivalents speeds woundhealing and prevents infection of the wound. In some preferredembodiments, the antimicrobial polypeptide is overexpressed in the humanskin equivalent is human beta defensins 1, 2, or 3 or combinationsthereof.

The present invention is not limited to the expression of any particularexogenous antimicrobial polypeptide in the human skin equivalents.Indeed, the expression of a variety of antimicrobial polypeptides iscontemplated, including, but not limited to the following: following:magainin (e.g., magainin I, magainin II, xenopsin, xenopsin precursorfragment, caerulein precursor fragment), magainin I and II analogs(PGLa, magainin A, magainin G, pexiganin, Z-12, pexigainin acetate, D35,MSI-78A, MG0 [K10E, K11E, F12W-magainin 2], MG2+ [K10E,F12W-magainin-2], MG4+ [F12W-magainin 2], MG6+[f12W, E19Q-magainin 2amide], MSI-238, reversed magainin II analogs [e.g., 53D, 87-ISM, andA87-ISM], Ala-magainin II amide, magainin II amide), cecropin P1,cecropin A, cecropin B, indolicidin, nisin, ranalexin, lactoferricin B,poly-L-lysine, cecropin A (1-8)-magainin II (1-12), cecropin A(1-8)-melittin (1-12), CA(1-13)-MA(1-13), CA(1-13)-ME(1-13), gramicidin,gramicidin A, gramicidin D, gramicidin S, alamethicin, protegrin,histatin, dermaseptin, lentivirus amphipathic peptide or analog, parasinI, lycotoxin I or II, globomycin, gramicidin S, surfactin, ralinomycin,valinomycin, polymyxin B, PM2 [(+/−) 1-(4-aminobutyl)-6-benzylindane],PM2c [(+/−)-6-benzyl-1-(3-carboxypropyl)indane], PM3[(+/−)1-benzyl-6-(4-aminobutyl)indane], tachyplesin, buforin I or II,misgurin, melittin, PR-39, PR-26, 9-phenylnonylamine, (KLAKKLA)n (SEQ IDNO: 124), (KLAKLAK)n (SEQ ID NO: 125), where n=1, 2, or 3, (KALKALK)n(SEQ ID NO: 126), (KLGKKLG)n (SEQ ID NO: 127), and (KAAKKAA)n (SEQ IDNO: 128), wherein n=1, 2, or 3, (paradaxin, Bac 5, Bac 7, ceratoxin,mdelin 1 and 5, bombin-like peptides, PGQ, cathelicidin, HD-5,Oabac5alpha, ChBac5, SMAP-29, Bac7.5, lactoferrin, granulysin, thionin,hevein and knottin-like peptides, MPG1, 1bAMP, snakin, lipid transferproteins, and plant defensins. Exemplary sequences for the abovecompounds are provided in Table 1. In some embodiments, theantimicrobial peptides are synthesized from L-amino acids, while inother embodiments, the peptides are synthesized from or comprise D-aminoacids.

In some preferred embodiments of the present invention, theantimicrobial polypeptide is a defensin. In certain embodiments, thedefensin comprises the following consensus sequence: (SEQ IDNO:107—X₁CN₁CRN₂CN₃ERN₄CN₅GN₆CCX₂, wherein N and X representconservatively or nonconservatively substituted amino acids and N₁=1,N₂=3 or 4, N₃=3 or 4, N₄=1, 2, or 3, N₆=5-9, X₁ and X₂ may be present,absent, or equal from 1-2.

In certain embodiments, mutant defensins are utilized in the methods andcompositions of the present invention. For example, in some embodiments,disulfide bond formation in beta-defensin 3 is disrupted by mutation ofone or more cysteine residues. In preferred embodiments, 5 of the 6cysteine residues (e.g., Cys₄₀, Cys₄₅, Cys₅₅, Cys₆₂, and Cys₆₃) aremutated to alanine or other uncharged amino acid not capable of formingdisulfide bonds. The present invention is not limited to a particularmechanism. Indeed, an understanding of the mechanism is not necessary topractice the present invention. Nonetheless, it is contemplated thatdisruption of disulfide bond formation in beta-defensin 3 increases theantimicrobial activity of the protein (See e.g., Hoover et al.,Antimicrobial agent and chemotherapy 47:2804 (2003) and Wu et al., PNAS100:8880 (2003)). The hBD-3 mutants of the present invention may havealtered (e.g., greater or less) antimicrobial activity than wild typehBD-3 or they may have similar antimicrobial activity. It is furthercontemplated that the disruption of disulfide bonds reduces oreliminates the ability of hBD-3 to elicit a chemotactic response. Theelimination of chemotactic response may be desirable for avoidance ofimmune response to skin equilavents grafted onto hosts (e.g., humanhosts).

In other embodiments, glycine to alanine substitutions are generated inhBD-3 (e.g., Gly38Ala). In some embodiments, the both Gly-Ala andCys-Ala substitutions are generated in the same hBD-3 polypeptide.

In some embodiments, antimicrobial polypeptides are modified to includea secretion signal peptide at the N-terminus of the antimicrobialpeptides to create a chimeric (hybrid) protein. It is contemplated thatsuch signal sequences allow for the free secretion of antimicrobialpeptides, rather than facilitating their association with the cellsurface. The antimicrobial peptides have an endogenous signal secretionpeptide that directs the immature peptide to the golgi apparatus andeventual secretion into intracellular spaces. These peptides appear tobe tightly associated with the cell surfaces, and not “freely” secreted.In some embodiments, the IL-2 Signal secretion peptide is used (CTT GCACTT GTC ACA AAC AGT GCA CCT; SEQ ID NO:108).

In other embodiments, the antimicrobial polypeptide is a humancathelicidin (hCAP18) polypeptide (SEQ ID NO:47).

The present invention is not limited to any particular antimicrobialpeptide. Indeed, media comprising a variety of antimicrobialpolypeptides are contemplated. Representative antimicrobial polypeptidesare provided in Table 1 below.

TABLE 1 Antimicrobial Peptides SEQ ID NO: Name Organism Sequence 13beta-defensin 1 Human MRTSYLLLFTLCLLLSEMASGGNFLTGLGHRSDHYNCVSSGGQCLYSACPIFTKIQGTCYRG KAKCCK 14 beta-defensin 2 HumanMRVLYLLFSFLFIFLMPLPGVFGGIGDPVTCL KSGAICHPVFCPRRYKQIGTCGLPGTKCCKK P 15beta-defensin 3 Human MRIHYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGGRCAVLSCLPKEEQIGKCSTRGRKCC RRKK 16 lingual antimicrobial Bos taurusmrlhhlllallflvlsagsgftqgvrnsqscrrnkgicvp peptide precursorircpgsmrqigtclgaqvkccrrk (Magainin) 17 antimicrobial peptideXenopus laevis Gvlsnvigylkklgtgalnavlkq PGQ 18 Xenopsin Xenopus laevismykgiflcvllavicanslatpssdadedndeveryvrgwaskigqtlgkiakvglkeliqpkreamlrsaeaqgkrpwil 19 magainin precursorXenopus laevis mfkglficsliavicanalpqpeasadedmderevrgigkflhsagkfgkafvgeimkskrdaeavgpeafadedlderevrgigkflhsakkfgkafvgeimnskrdaeavgpeafadedlderevrgigkflhsakkfgkafvgeimnskrdaeavgpeafadedlderevrgigkflhsakkfgkafvgeimnskrdaeavgpeafadedfderevrgigkflhsakkfgkafvgeimnskrdaeavgpeafadedlderevrgigkflhsakkfgk afvgeimnskrdaeavddrrwve 20tachyplesin I Tachypleus kwcfrvcyrgicyrrcr gigas 21 tachyplesin IITachypleus rwcfrvcyrgicyrkcr gigas 22 buforin I Bufo bufomsgrgkqggkvrakaktrssraglqfpvgrvhrllrkgny gagarizansaqrvgagapvylaavleyltaeilelagnaardnkktriiprhlqlavrndeelnkllggvtiaqggvlpniqavllpkt esskpaksk 23 buforin IIBufo bufo trssraglqfpvgrvhrllrk gagarizans 24 cecropin A Bombyx morimnfvrilsfvfalvlalgaysaapeprwklfkkiekvgrn vrdglikagpaiavigqakslgk 25cecropin B Bombyx mori mnfakilsfvfalvlalsmtsaapeprwkifkkiekmgrnirdgivkagpaievlgsakaigk 26 cecropin C Drosophilamnfykifvfvalilaisigqseagwlkklgkrierigqht melanogasterrdatiqglgiaqqaanvaatarg 27 cecropin P1 Sus scrofaswlsktakklensakkrisegiaiaiqggpr 28 Indolicidin Bos taurus ilpwkwpwwpwrr29 Nisin Lactococcus itsislctpgcktgalmgcnmktatchcsihvsk lactis 30Ranalexin Rana flgglikivpamicavtkkc catesbeiana 31 lactoferricin BBos taurus fkcrrwqwrmkklgapsitcvrraf 32 Protegrin-1 Sus scrofarggrlcycrrrfcvcvgrx 33 Protegrin-2 Sus scrofa ggrlcycrrrfcicvg 34histatin precursor Homo sapiens mkffvfalilalmlsmtgadshakrhhgykrkfhekhhshrgyrsnylydn 35 histatin 1 Macaca dsheerhhgrhghhkygrkfhekhhshrgyrsnylydnfascicularis 36 dermaseptin Phyllomedusaalwktmlkklgtmalhagkaalgaaadtisqtq sauvagei 37 dermaseptin 2 Phyllomedusaalwftmlkklgtmalhagkaalgaaantisqgtq sauvagei 38 dermaseptin 3Phyllomedusa alwknmlkgigklagkaalgavkklvgaes sauvagei 39 MisgurinMisgurnus rqrveelskfskkgaaarrrk anguillicaudatus 40 MelittinApis mellifera gigavlkvlttglpaliswisrkkrqq 41 pardaxin-1 Pardachirusgffalipkiissplfktllsavgsalsssgeqe pavoninus 42 pardaxin-2 Pardachirusgffalipkiisspifktllsavgsalsssggqe pavoninus 43 Bactenecin 5 precursorBos taurus metqraslslgrcslwllllglvlpsasaqalsyreavlravdqfnersseanlyrlleldptpnddldpgtrkpvsfrvketdcprtsqqpleqcdfkenglvkqcvgtvtldpsndqfdincnelqsvrfrppirrppirppfyppfrppirppifpp irppfrpplgpfpgrr 44bactenecin precursor Bos taurus metpraslslgrwslwlllglalpsasaqalsyreavlravdqlneqssepniyrlleldqppqddedpdspkrvsfrvketvcsrttqqppeqcdfkenglllrcegtvtldqvrgnfditcnnhqsiritkqpwappqaarlcrivvirvcr 45 ceratotoxin A Ceratitissigsalkkalpvakkigkialpiakaalp capitata 46 ceratotoxin B Ceratitissigsafkkalpvakkigkaalpiakaalp capitata 47 cathelicidin antimicrobialHomo sapiens mktqrnghslgrwslvllllglvmplaiiaqvlsykeavl peptideraidginqrssdanlyrlldldprptmdgdpdtpkpvsftvketvcprttqqspedcdfkkdglvkrcmgtvtlnqargsfdiscdkdnkrfallgdffrkskekigkefkrivqrikdf lrnlvprtes 48myeloid cathelicidin 3 Equus caballusmetqrntrclgrwspllllglvippattqalsykeavlravdglnqrssdenlyrlleldplpkgdkdsdtpkpvsfmvketvcprimkqtpeqcdfkenglvkqcvgtvildpvkdyfdascdepqrvkrfhsvgsliqrhqqmirdkseatrhgiri itrpklllas 49myeloid antimicrobial Bos taurusmetqraslslgrwslwllllglalpsasaqalsyreavlr peptide BMAP-28avdqlneksseanlyrlleldpppkeddenpnipkpvsfrvketvcprtsqqspeqcdfkengllkecvgtvtldqvgsnfditcavpqsvgglrslgrkilrawkkygpiivpiirig 50 myeloid cathelicidin 1Equus caballus metqrntrclgrwsplllllglvippattqalsykeavlravdglnqrssdenlyrlleldplpkgdkdsdtpkpvsfmvketvcprimkqtpeqcdfkenglvkqcvgtvilgpvkdhfdvscgepqrvkrfgrlaksflrmrillprrkillas 51 SMAP 29 Ovis ariesmetqraslslgrcslwllllglalpsasaqvlsyreavlraadqlneksseanlyrlleldpppkqddensnipkpvsfrvketvcprtsqqpaeqcdfkengllkecvgtvtldqvrnnfditcaepqsvrghrlgrkiahgvkkygptvlriiriag 52 BNP-1 Bos taurus rlcrivvirvcr53 HNP-1 Homo sapiens acycripaciagerrygtciyqgrlwafcc 54 HNP-2Homo sapiens cycripaciagerrygtciyqgrlwafcc 55 HNP-3 Homo sapiensdcycripaciagerrygtciyqgrlwafcc 56 HNP-4 Homo sapiensvcscrlvfcrrtelrvgncliggvsftycctry 57 NP-1 Oryctolagusvvcacrralclprerragfcrirgrihplccrr cuniculus 58 NP-2 Oryctolagusvvcacrralclplerragfcrirgrihplccrr cuniculus 59 NP-3A Oryctolagusgicacrrrfcpnserfsgycrvngaryvrccsrr cuniculus 60 NP-3B Oryctolagusgrcvcrkqllcsyrerrigdckirgvrfpfccpr cuniculus 61 NP-4 Oryctolagusvsctcrrfscgfgerasgsctvnggvrhtlccrr cuniculus 62 NP-5 Oryctolagusvfctcrgflcgsgerasgsctingvrhtlccrr cuniculus 63 RatNP-1 Rattusvtcycrrtrcgfrerlsgacgyrgriyrlccr norvegicus 64 Rat-NP-3 Rattuscscrysscrfgerllsgacringriyrlcc norvegicus 65 Rat-NP-4 Rattusactcrigacvsgerltgacglngriyrlccr norvegicus 66 GPNP Guinea pigrrcicttrtcrfpyrrlgtcifqrn-vytfcc 67 theta defensin-1 Macacarcictrgfcrcicrrgvc mulatta 68 defensin CUA1 Helianthusmkssmkmfaalllvvmcllanemggplvveartcesqshk annuusfkgtclsdtncanvchserfsggkcrgfrrrcfctthc 69 defensin SD2 Helianthusmkssmkmfaalllvvmcllanemggplvveartcesqshk annuusfkgtclsdincanvchserfsggkcrgfrrrcfctthc 70 neutrophil defensin 2 Macacaacycripaclagerrygtcfymgrvwafcc mulatta 71 4 KDA defensin Androctonusgfgcpfnqgachrhcrsirrrggycaglfkqtctcyr australis hector 72 defensinMytilus gfgcpnnyqchrhcksipgreggyeggxhrlrctcyrc galloprovincialis 73defensin AMP1 Heuchera dgvklcdvpsgtwsghcgssskcsqqckdrehfayggachsanguinea yqfpsvkcfckrqc 74 defensin AMP1 Clitorianlcerasltwtgncgntghcdtqcrnwesakhgachkrgn ternatea wkcfcyfnc 75cysteine-rich cryptdin-1 Mus musculusmkklvllfalvllafqvqadsiqntdeetkteeqpgekdq homologavsvsfgdpqgsalqdaalgwgrrcpqcprcpscpscprc prcprckcnpk 76 beta-defensin-9Bos taurus qgvrnfvtcrinrgfcvpircpghrrqigtclgpqikccr 77 beta-defensin-7Bos taurus qgvrnfvtcrinrgfcvpircpghrrqigtclgprikccr 78 beta-defensin-6Bos taurus qgvrnhvtcriyggfcvpircpgrtrqigtcfgrpvkccrrw 79 beta-defensin-5Bos taurus qvvrnpqscrwnmgvcipiscpgnmrqigtcfgprvpccr 80 beta-defensin-4Bos taurus qrvrnpqscrwnmgvcipflcrvgmrqigtcfgprvpccrr 81 beta-defensin-3Bos taurus qgvrnhvtcrinrgfcvpircpgrtrqigtcfgprikccrsw 82beta-defensin-10 Bos taurus qgvrsylscwgnrgicllnrcpgrmrqigtclaprvkccr 83beta-defensin-13 Bos taurus sgisgplscgrnggvcipircpvpmrqigtcfgrpvkccrsw84 beta-defensin-1 Bos taurus dfaschtnggiclpnrcpghmiqigicfrprvkccrsw 85coleoptericin Zophobas slqggapnfpqpsqqnggwqvspdlgrddkgntrgqieiq atratusnkgkdhdfnagwgkvirgpnkakptwhvggtyrr 86 defensin C Aedes aegyptiatcdllsgfgvgdsacaahciargnrggycnskkvcvcrn 87 defensin B Mytilus edulisgfgcpndypchrhcksipgryggyeggxlnirctc 88 sapecin C Sarcophagaatcdllsgigvqhsacalhcvfrgnrggyctgkgicvcrn peregrina 89macrophage antibiotic Oryctolagusmrtlallaaillvalqaqaehvsysidevvdqqppqaedq peptide MCP-1 cuniculusdvaiyvkehessalealgvkagvvcacrralclprerrag fcrirgrihplccrr 90 cryptdin-2Mus musculus mkplvllsalvllsfqvqadpiqntdeetkteeqsgeedqavsvsfgdregaslqeeslrdlvcycrtrgckrrermngt crkghlmytlcc 91 cryptdin-5Mus musculus mktfvllsalvllafqvqadpihktdeetnteeqpgeedqaysisfggqegsalheelskklicycrirgclurervfgt crnlfltfvfccs 92 cryptdin 12Mus musculus 1rdlycycrargckgrermngtcrkghllymlccr 93 defensin Pyrrhocorisatcdilsfqsqwvtpnhagcalhcvikgykggqckitvchcrr apterus 94 defensin R-5Rattus vtcycrstrcgfrerlsgacgyrgriyrlccr norvegicus 95 defensin R-2Rattus vtcscrtsscrfgerlsgacringriyncc norvegicus 96 defensin NP-6Oryctolagus gicacrrrfclnfeqfsgyervngaryvrccsn cuniculus 97beta-defensin-2 Pan troglodytes mrvlyllfsflfiflmplpgvfggisdpvtclksgaichpvfcprrykqigtcglpgtkcckkp 98 beta-defensin-1 Capra hircusmrlhhlllvlfflvlsagsgftqgirsrrschrnkgvcal trcprnmrqigtcfgppvkccrkk 99beta defensin-2 Capra hircus mrlhhlllalfflvlsagsgftqgiinhrscyrnkgvcaparcprnmrqigtchgppvkccrkk 100 defensin-3 Macacamrtivilaaillvalqaqaeplqartdeataaqeqiptdn mulattapevvvslawdeslapkdsvpglrknmacycripaclager rygtcfyrrrvwafcc 101 defensin-1Macaca mrtivilaaillvalqaqaeplqartdeataaqeqiptdn mulattapevvvslawdeslapkdsvpglrknmacycripaclager rygtcfylgrvwafcc 102neutrophil defensin 1 Mesocricetus vtcfcrn-gcasrerhigyerfgntiyrlcerrauratus 103 neutrophil defensin 1 Mesocricetuscfckrpvcdsgetqigycrlgntfyrlccrq auratus 104 Gallinacin 1-alphaGallus gallus grksdcfrkngfcaflkcpyltlisgkcsrfhlcckriw 105 defensinAllomyrina vtcdllsfeakgfaanhslcaahclaigrrggscergvcicrr dichotoma 106neutrophil cationic Cavia porcellus rrcicttrtcrfpyrrlgtcifqnrvytfccpeptide 1

Accordingly, in some embodiments the present invention contemplates theproduction of keratinocytes and skin equivalents expressing anantimicrobial polypeptide, and compositions and methods for makingkeratinocytes expressing an exogenous antimicrobial polypeptide. Inpreferred embodiments, the antimicrobial polypeptide is a defensin or acathelicidin. In still more preferred embodiments, the defensin is ahuman beta defensin. In still more preferred embodiments, the human betadefensin is human beta defensin 1, 2 or 3. In some embodiments, thekeratinocytes are transfected with more than one defensin selected fromthe group consisting of human beta-defensin 1, 2 or 3. In preferredembodiments, keratinocytes are induced to express an antimicrobialpolypeptide through transfection with an expression vector comprising agene encoding an antimicrobial polypeptide. An expression vectorcomprising a gene encoding an antimicrobial polypeptide can be producedby operably linking an antimicrobial polypeptide coding sequence to oneor more regulatory sequences such that the resulting vector is operablein a desired host.

In preferred embodiments, the antimicrobial polypeptide is isolated froma DNA source, cloned, sequenced, and incorporated into a selectionvector. In certain embodiments, isolation of the antimicrobialpolypeptide DNA occurs via PCR by using primer sequences designed toamplify the antimicrobial polypeptide sequence. Primer sequencesspecific for the desired antimicrobial polypeptide may be obtained fromGenbank. Amplification of a DNA source with such primer sequencesthrough standard PCR procedures results in antimicrobial polypeptidecDNA isolation. In preferred embodiments, the source of cDNA is humancDNA.

D) Methods of Generating Host Cells Expressing Exogenous Polypeptides

In some embodiments, the present invention provides methods ofgenerating host cells (e.g., keratinocytes) and skin equivalentsexpressing one or more exogenous polypeptides (e.g., KGF-2 and/orantimicrobial polypeptides. The present invention is not limited toparticular methods for the generation of such cells and skinequivalents. Exemplary methods are described below. Additional methodsare known to those skilled in the relevant arts.

In certain embodiments, the antimicrobial polypeptide cDNA is clonedinto a cloning vector. A regulatory sequence that can be linked to theantimicrobial polypeptide DNA sequence in an expression vector is apromoter that is operable in the host cell in which the antimicrobialpolypeptide is to be expressed. Optionally, other regulatory sequencescan be used herein, such as one or more of an enhancer sequence, anintron with functional splice donor and acceptance sites, a signalsequence for directing secretion of the defensin, a polyadenylationsequence, other transcription terminator sequences, and a sequencehomologous to the host cell genome. Other sequences, such as origin ofreplication, can be added to the vector as well to optimize expressionof the desired defensin. Further, a selectable marker can be present inthe expression vector for selection of the presence thereof in thetransformed host cells.

In preferred embodiments, antimicrobial polypeptide is fused to aregulatory sequence that drives the expression of the polypeptide (e.g.,a promoter). In preferred embodiments, the regulatory sequence is theinvolucrin promoter (SEQ ID NO: 12) or the keratin-14 promoter. However,any promoter that would allow expression of the antimicrobialpolypeptide in a desired host can be used in the present invention.Mammalian promoter sequences that can be used herein are those frommammalian viruses that are highly expressed and that have a broad hostrange. Examples include the SV40 early promoter, the Cytomegalovirus(“CMV”) immediate early promoter mouse mammary tumor virus long terminalrepeat (“LTR”) promoter, adenovirus major late promoter (Ad MLP), andHerpes Simplex Virus (“HSV”) promoter. In addition, promoter sequencesderived from non-viral genes, such as the murine metallothionein gene,ubiquitin and elongation factor alpha (EF-1α) are also useful herein.These promoters can further be either constitutive or regulated, such asthose that can be induced with glucocorticoids in hormone-responsivecells.

In some preferred embodiments, host cells (e.g., keratinocytes cells)expressing KGF-2 or antimicrobial polypeptides can be produced byconventional gene expression technology, as discussed in more detailbelow. The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, recombinant DNA, and immunology, which are within theskill of the art. Such techniques are explained fully in the literature,including Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nded. (Cold Spring Harbor Laboratory Press, 1989); DNA CLONING, Vol. I andII, D. N Glover ed. (IRL Press, 1985); OLIGONUCLEOTIDE SYNTHESIS, M. J.Gait ed. (IRL Press, 1984); NUCLEIC ACID HYBRIDIZATION, B. D. Hames & S.J. Higgins eds. (IRL Press, 1984); TRANSCRIPTION AND TRANSLATION, B. D.Hames & S. J. Higgins eds., (IRL Press, 1984); ANIMAL CELL CULTURE, R.I. Freshney ed. (IRL Press, 1986); IMMOBILIZED CELLS AND ENZYMES, K.Mosbach (IRL Press, 1986); B. Perbal, A PRACTICAL GUIDE TO MOLECULARCLONING, Wiley (1984); the series, METHODS IN ENZYMOLOGY, AcademicPress, Inc.; GENE TRANSFER VECTORS FOR MAMMALIAN CELLS, J. H. Miller andM. P. Calos eds. (Cold Spring Harbor Laboratory, 1987); METHODS INENZYMOLOGY, Vol. 154 and 155, Wu and Grossman, eds., and Wu, ed.,respectively (Academic Press, 1987), IMMUNOCHEMICAL METHODS IN CELL ANDMOLECULAR BIOLOGY, R. J. Mayer and J. H. Walker, eds. (Academic PressLondon, Harcourt Brace U.S., 1987), PROTEIN PURIFICATION: PRINCIPLES ANDPRACTICE, 2nd ed. (Springer-Verlag, N.Y. (1987), and HANDBOOK OFEXPERIMENTAL IMMUNOLOGY, Vol. I-IV, D. M. Weir et al., (BlackwellScientific Publications, 1986); Kitts et al., Biotechniques 14:810-817(1993); Munemitsu et al., Mol. and Cell. Biol. 10:5977-5982 (1990).

The present invention contemplates keratinocytes and skin equivalentsexpressing KGF-2 and/or antimicrobial polypeptides, and compositions andmethods for making such cells. In some embodiments, host cells areinduced to express exogenous polypeptides through transfection with anexpression vector containing DNA encoding the exogenous polypeptide. Anexpression vector containing KGF-2 DNA can be produced by operablylinking KGF-2 to one or more regulatory sequences such that theresulting vector is operable in a desired host. Cell transformationprocedures suitable for use herein are those known in the art andinclude, for example with mammalian cell systems, dextran-mediatedtransfection, calcium phosphate precipitation, polybrene-mediatedtransfection, protoplast fusion, electroporation, encapsulation of theexogenous polynucleotide in liposomes, and direct microinjection of theDNA into nuclei. In preferred embodiments, cells are transfected with apUB-Bsd expression vector containing exogenous DNA (e.g., KGF-2 andantimicrobial polypeptides) operably linked to promoter (e.g., K14 orinvolucrin) DNA.

Immunoassays and activity assays that are known in the art can beutilized herein to determine if the transformed host cells areexpressing the desired exogenous polypeptide (e.g., KGF-2 andantimicrobial polypeptides). In some embodiments, detection ofintracellular production of KGF-2 or antimicrobial polypeptides bytransformed host cells is accomplished with an immunofluorescence assay.In preferred embodiments, detection of intracellular production ofexogenous polypeptides by transformed host cells is accomplished througha RT-PCR screen. In further embodiments, detection of secreted orextracellular production of KGF-2 or antimicrobial polypeptides bytransformed host cells is accomplished through a direct ELISA screen. Insome embodiments, the KGF-2 or antimicrobial polypeptide is detected byWestern blotting.

In other embodiments, expression vectors comprising exogenouspolypeptides are introduced directly into tissues (e.g., human skinequivalents). Expression vectors may be introduced into tissues usingany suitable technique including, but not limited to, electroporation,particle bombardment (e.g., 6,685,669, 6,592,545, and 6,004,286; each ofwhich is herein incorporated by reference) and transfection.

II. Selection of Cells by Electroporation

Experiments conducted during the course of development of the presentinvention (See e.g., Example 26) resulted in the identification of anovel technique for the selection of cells within a population. Theexperiments demonstrated that cells electroporated in the presence orabsence of exogenous nucleic acid and selection demonstrated propertiesof multipotency. Accordingly, in some embodiments, the present inventionprovides methods of selecting for cells in a population having desiredgrowth and proliferation properties.

In some embodiments, electroporation is used to select for cells withenhanced pluripotency or multipotency. In other embodiments,electroporation is used to select for cells with enhanced pluripotencyor multipotency. As used herein, the term “pluripotent” means theability of a cell to differentiate into the three main germ layers:endoderm, ectoderm, and mesoderm. In some embodiments, the cells withenhanced pluripotency or multipotency exhibit stem cells likeproperties.

For example, in some embodiments, electroporation is used to select forcells with stem-cell like properties. Stem cells are undifferentiatedcells that can give rise to a succession of mature functional cells.Stem cells can by embryonically derived (See e.g., U.S. Pat. Nos.5,843,780 and 6,200,806; each of which is herein incorporated byreference) or derived from adult cells. Examples of adult stem cellsinclude hematopoietic stem cells, neural stem cells, mesenchymal stemcells, and bone marrow stromal cells. These stem cells have demonstratedthe ability to differentiate into a variety of cell types includingadipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromalcells, and thymic stroma (mesenchymal stem cells); hepatocytes, vascularcells, and muscle cells (hematopoietic stem cells); myocytes,hepatocytes, and glial cells (bone marrow stromal cells) and, cells fromall three germ layers (adult neural stem cells).

In other embodiments, electroporation is used to select for cells withextended proliferative capacity. For example, experiments conductedduring the course of development of the present invention demonstratedthat electroporated cells were typically the larger surviving colonies.

In yet other embodiments, electroporation is used to select forkeratinocytes having holoclone or meroclone cell morphology (e.g., acolony morphology of tightly packed, uniform cells, smooth colony edges,overall round colony morphology).

III. Treatment of Wounds with Keratinocytes Cells Transfected withExogenous Polypeptides

Successful treatment of chronic skin wounds (e.g., venous ulcers,diabetic ulcers, pressure ulcers) is a serious problem. The healing ofsuch a wound often times takes well over a year of treatment. Treatmentoptions currently include dressings and debridement (use of chemicals orsurgery to clear away necrotic tissue), and/or antibiotics in the caseof infection. These treatment options take extended periods of time andhigh amounts of patient compliance. As such, a therapy that can increasea practioner's success in healing chronic wounds and accelerate the rateof wound healing would meet an unmet need in the field.

In some embodiments, the present invention contemplates treatment ofskin wound with keratinocytes and skin equivalents expression exogenousantimicriobial and/or KGF-2 polypeptides.

KGF-2 is associated with skin wound healing. In skin, KGF-2 is naturallyexpressed in the dermal compartment. Topical application of KGF-2 toskin wounds increases dermal cell proliferation. In addition, KGF-2manifests strong mitogenic activity in dermal cells and stimulatesgranulation tissue formation in full thickness excisional wounds. KGF-2accelerated wound closure is transient and does not cause scar formationafter complete wound healing (Yu-Ping et al. 1999). Local proteinadministration, however, has been shown to be ineffective due to enzymesand proteases in the wound fluid (Jeschke et al. 2002). KGF-2selectively induces normal epithelial cell proliferation,differentiation and migration, while having no in vitro or in vivoproliferative effects on KGFR (+) human epithelial-like tumors.(Alderson et al. 2002). As such, KGF-2 is an attractive candidate fortherapeutic use to enhance wound healing.

The present invention contemplates treatment of skin wounds withkeratinocytes or skin equivalents expressing KGF-2 and/or antimicrobialpolypeptides. In some embodiments, cells expressing KGF-2 and/orantimicrobial polypeptides are topically applied to wound sites. In someembodiments, the keratinocytes are applied via a spray, while in otherembodiments, the keratinocytes are applied via a gel. In otherembodiments, cells expressing KGF-2 and/or antimicrobial polypeptidesare used for engraftment on partial thickness wounds. In otherembodiments, cells expressing KGF-2 and/or antimicrobial polypeptidesare used for engraftment on full thickness wounds. In other embodiments,cells expressing KGF-2 and/or antimicrobial polypeptides are used totreat numerous types of internal wounds, including, but not limited to,internal wounds of the mucous membranes that line the gastrointestinaltract, ulcerative colitis, and inflammation of mucous membranes that maybe caused by cancer therapies. In still other embodiments, cellsexpressing KGF-2 and/or antimicrobial polypeptides are used as atemporary or permanent wound dressing.

Cells expressing KGF-2 and/or antimicrobial polypeptides find use inwound closure and burn treatment applications. The use of autografts andallografts for the treatment of burns and wound closure is described inMyers et al., A. J. Surg. 170(1):75-83 (1995) and U.S. Pat. Nos.5,693,332; 5,658,331; and 6,039,760, each of which is incorporatedherein by reference. In some embodiments, the skin equivalents may beused in conjunction with dermal replacements such as DERMAGRAFT. Inother embodiments, the skin equivalents are produced using both astandard source of keratinocytes (e.g., NIKS cells) and keratinocytesfrom the patient that will receive the graft. Therefore, the skinequivalent contains keratinocytes from two different sources. In stillfurther embodiments, the skin equivalent contains keratinocytes from ahuman tissue isolate. Accordingly, the present invention providesmethods for wound closure, including wounds caused by burns, comprisingproviding cells expressing KGF-2 and/or antimicrobial polypeptides and apatient suffering from a wound and treating the patient with the cellsunder conditions such that the wound is closed.

Detailed methods for producing the skin equivalents of the presentinvention are disclosed in the following Experimental section. However,the present invention is not limited to the production of skinequivalents by the methods. Indeed, a variety of organotypic culturetechniques may be used to produce skin equivalents, including thosedescribed in U.S. Pat. Nos. 5,536,656 and 4,485,096, both of which areincorporated herein by reference. In some embodiments, differentpopulations of keratinocytes are used to construct the skin equivalent.Accordingly, in some embodiments, the skin equivalents of the presentinvention are formed from keratinocytes derived from an immortalizedcell line (e.g., NIKS cells) and cell derived from a patient. In otherembodiments, the skin equivalents of the present invention are formedfrom at least a first population of keratinocytes derived from animmortalized cell line that express a exogenous antimicrobialpolypeptide and/or KGF-2 and a second population of keratinocytesderived from an immortalized cell line that do not express a exogenousantimicrobial polypeptide. It is contemplated that varying the ratio ofthe two populations the dose of antimicrobial polypeptide and/or KGF-2delivered can be varied. In still other embodiments, the skinequivalents are formed from at least a first population of keratinocytesexpressing a first exogenous antimicrobial polypeptide (e.g., hBD-1) andat least a second population of keratinocytes expressing a secondexogenous antimicrobial polypeptide (e.g., hBD-2 or hBD-3). Again, theratios of the cell populations can be varied to vary the dose. In stillother embodiments, the skin equivalents are formed from at least a firstpopulation of keratinocytes expressing a first exogenous antimicrobialpolypeptide (e.g., hBD-1), at least a second population of keratinocytesexpressing a second exogenous antimicrobial polypeptide (e.g., hBD-2 orhBD-3), and keratinocytes derived from a patient.

In a further embodiment, the KGF-2 and/or antimicrobial polypeptide or aconjugate thereof can be mixed with a pharmaceutically acceptablecarrier to produce a therapeutic composition that can be administeredfor therapeutic purposes, for example, for wound healing, and fortreatment of hyperproliferative diseases of the skin and tumors, such aspsoriasis and basal cell carcinoma.

In still further embodiments, the cells expressing KGF-2 and/orantimicrobial polypeptides are engineered to provide a therapeutic agentto a subject. The present invention is not limited to the delivery ofany particular therapeutic agent. Indeed, it is contemplated that avariety of therapeutic agents may be delivered to the subject,including, but not limited to, enzymes, peptides, peptide hormones,other proteins, ribosomal RNA, ribozymes, and antisense RNA. Thesetherapeutic agents may be delivered for a variety of purposes, includingbut not limited to the purpose of correcting genetic defects. In someparticular preferred embodiments, the therapeutic agent is delivered forthe purpose of detoxifying a patient with an inherited inborn error ofmetabolism (e.g., aminoacidopathesis) in which the graft serves aswild-type tissue. It is contemplated that delivery of the therapeuticagent corrects the defect. In some embodiments, the cells expressingKGF-2 and/or antimicrobial polypeptides are transfected with a DNAconstruct encoding a therapeutic agent (e.g., insulin, clotting factorIX, erythropoietin, etc) and the cells grafted onto the subject. Thetherapeutic agent is then delivered to the patient's bloodstream orother tissues from the graft. In preferred embodiments, the nucleic acidencoding the therapeutic agent is operably linked to a suitablepromoter. The present invention is not limited to the use of anyparticular promoter. Indeed, the use of a variety of promoters iscontemplated, including, but not limited to, inducible, constitutive,tissue specific, and keratinocyte specific promoters. In someembodiments, the nucleic acid encoding the therapeutic agent isintroduced directly into the keratinocytes (i.e., by calcium phosphateco-precipitation or via liposome transfection). In other preferredembodiments, the nucleic acid encoding the therapeutic agent is providedas a vector and the vector is introduced into the keratinocytes bymethods known in the art. In some embodiments, the vector is an episomalvector such as a plasmid. In other embodiments, the vector integratesinto the genome of the keratinocytes. Examples of integrating vectorsinclude, but are not limited to, retroviral vectors, adeno-associatedvirus vectors, and transposon vectors.

IV. Testing Methods

The host cells and cultured skin tissue of the present invention may beused for a variety of in vitro tests. In particular, the host cells andcultured skin tissue find use in the evaluation of: skin care products,drug metabolism, cellular responses to test compounds, wound healing,phototoxicity, dermal irritation, dermal inflammation, skin corrosivity,and cell damage. The host cells and cultured skin tissue are provided ina variety of formats for testing, including 6-well, 24-well, and 96-wellplates. Additionally, the cultured skin tissue can be divided bystandard dissection techniques and then tested. The cultured skin tissueof the present invention may have both an epidermal layer with adifferentiated stratum corneum and dermal layer that includes dermalfibroblasts. As described above, in preferred embodiments, the epidermallayer is derived from immortalized NIKS cells. Other preferred celllines, including NIKS cells are characterized by; i) being immortalized;ii) being nontumorigenic; iii) forming cornified envelopes when inducedto differentiate; iv) undergoing normal squamous differentiation inorganotypic culture; and v) maintaining cell type-specific growthrequirements, wherein said cell type-specific growth requirementsinclude 1) exhibition of morphological characteristics of normal humankeratinocytes when cultured in standard keratinocyte growth medium inthe presence of mitomycin C-treated 3T3 feeder cells; 2) dependence onepidermal growth factor for growth; and 3) inhibition of growth bytransforming growth factor β1.

The present invention encompasses a variety of screening assays. In someembodiments, the screening method comprises providing a host cell orcultured skin tissue of the present invention and at least one testcompound or product (e.g., a skin care product such as a moisturizer,cosmetic, dye, or fragrance; the products can be in any from, including,but not limited to, creams, lotions, liquids and sprays), applying theproduct or test compound to the host cell or cultured skin tissue, andassaying the effect of the product or test compound on the host cell orcultured skin tissue. A wide variety of assays are used to determine theeffect of the product or test compound on the cultured skin tissue.These assays include, but are not limited to, MTT cytotoxicity assays(Gay, The Living Skin Equivalent as an In Vitro Model for Ranking theToxic Potential of Dermal Irritants, Toxic. In Vitro (1992)) and ELISAto assay the release of inflammatory modulators (e.g., prostaglandin E2,prostacyclin, and interleukin-1-alpha) and chemoattractants. The assayscan be further directed to the toxicity, potency, or efficacy of thecompound or product. Additionally, the effect of the compound or producton growth, barrier function, or tissue strength can be tested.

In particular, the present invention contemplates the use of host cellsor cultured skin tissue for high throughput screening of compounds fromcombinatorial libraries (e.g., libraries containing greater than 10⁴compounds). In some embodiments, the cells are used in second messengerassays that monitor signal transduction following activation ofcell-surface receptors. In other embodiments, the cells can be used inreporter gene assays that monitor cellular responses at thetranscription/translation level. In still further embodiments, the cellscan be used in cell proliferation assays to monitor the overallgrowth/no growth response of cells to external stimuli.

In second messenger assays, host cells or cultured skin tissue istreated with a compound or plurality of compounds (e.g., from acombinatorial library) and assayed for the presence or absence of asecond messenger response. In some preferred embodiments, the cells(e.g., NIKS cells) used to create cultured skin tissue are transfectedwith an expression vector encoding a recombinant cell surface receptor,ion-channel, voltage gated channel or some other protein of interestinvolved in a signaling cascade. It is contemplated that at least someof the compounds in the combinatorial library can serve as agonists,antagonists, activators, or inhibitors of the protein or proteinsencoded by the vectors. It is also contemplated that at least some ofthe compounds in the combinatorial library can serve as agonists,antagonists, activators, or inhibitors of protein acting upstream ordownstream of the protein encoded by the vector in a signal transductionpathway.

In some embodiments, the second messenger assays measure fluorescentsignals from reporter molecules that respond to intracellular changes(e.g., Ca²⁺ concentration, membrane potential, pH, IP3, cAMP,arachidonic acid release) due to stimulation of membrane receptors andion channels (e.g., ligand gated ion channels; see Denyer et al., DrugDiscov. Today 3:323-32 [1998]; and Gonzales et al., Drug. Discov. Today4:431-39 [1999]). Examples of reporter molecules include, but are notlimited to, FRET (florescence resonance energy transfer) systems (e.g.,Cuo-lipids and oxonols, EDAN/DABCYL), calcium sensitive indicators(e.g., Fluo-3, FURA 2, INDO 1, and FLUO3/AM, BAPTA AM),chloride-sensitive indicators (e.g., SPQ, SPA), potassium-sensitiveindicators (e.g., PBFI), sodium-sensitive indicators (e.g., SBFI), andpH sensitive indicators (e.g., BCECF).

In general, the cells comprising cultured skin tissue are loaded withthe indicator prior to exposure to the compound. Responses of the hostcells to treatment with the compounds can be detected by methods knownin the art, including, but not limited to, fluorescence microscopy,confocal microscopy (e.g., FCS systems), flow cytometry, microfluidicdevices, FLIPR systems (See, e.g., Schroeder and Neagle, J. Biomol.Screening 1:75-80 [1996]), and plate-reading systems. In some preferredembodiments, the response (e.g., increase in fluorescent intensity)caused by compound of unknown activity is compared to the responsegenerated by a known agonist and expressed as a percentage of themaximal response of the known agonist. The maximum response caused by aknown agonist is defined as a 100% response. Likewise, the maximalresponse recorded after addition of an agonist to a sample containing aknown or test antagonist is detectably lower than the 100% response.

The host cells and cultured skin tissue of the present invention arealso useful in reporter gene assays. Reporter gene assays involve theuse of host cells transfected with vectors encoding a nucleic acidcomprising transcriptional control elements of a target gene (i.e., agene that controls the biological expression and function of a diseasetarget or inflammatory response) spliced to a coding sequence for areporter gene. Therefore, activation of the target gene results inactivation of the reporter gene product. This serves as indicator ofresponse such an inflammatory response. Therefore, in some embodiments,the reporter gene construct comprises the 5′ regulatory region (e.g.,promoters and/or enhancers) of a protein that is induced due to skininflammation or irritation or protein that is involved in the synthesisof compounds produced in response to inflammation or irritation (e.g.,prostaglandin or prostacyclin) operably linked to a reporter gene.Examples of reporter genes finding use in the present invention include,but are not limited to, chloramphenicol transferase, alkalinephosphatase, firefly and bacterial luciferases, β-galactosidase,β-lactamase, and green fluorescent protein. The production of theseproteins, with the exception of green fluorescent protein, is detectedthrough the use of chemiluminescent, colorimetric, or bioluminescentproducts of specific substrates (e.g., X-gal and luciferin). Comparisonsbetween compounds of known and unknown activities may be conducted asdescribed above.

In other preferred embodiments, the host cells or cultured skin tissuefind use for screening the efficacy of drug introduction across the skinor the affect of drugs directed to the skin. In these embodiments,cultured skin tissue or host cells are treated with the drug deliverysystem or drug, and the permeation, penetration, or retention or thedrug into the skin equivalent is assayed. Methods for assaying drugpermeation are provided in Asbill et al., Pharm Res. 17(9): 1092-97(2000). In some embodiments, cultured skin tissue is mounted on top ofmodified Franz diffusion cells. The cultured skin tissue is allowed tohydrate for one hour and then pretreated for one hour with propyleneglycol. A saturated suspension of the model drug in propylene glycol isthen added to the cultured skin tissue. The cultured skin tissue canthen be sampled at predetermined intervals. The cultured skin tissue isthen analyzed by HPLC to determine the concentration of the drug in thesample. Log P values for the drugs can be determined using the ACDprogram (Advanced Chemistry Inc., Ontario, Canada). These methods may beadapted to study the delivery of drugs via transdermal patches or otherdelivery modes.

It is contemplated that cultured skin tissue of the present invention isalso useful for the culture and study of tumors that occur naturally inthe skin as well as for the culture and study of pathogens that affectthe skin. Accordingly, in some embodiments, it contemplated that thecultured skin tissue of the present invention is seeded with malignantcells. By way of non-limiting example, the cultured skin tissue can beseeded with malignant SCC13y cells as described in U.S. Pat. No.5,989,837, which is incorporated herein by reference, to provide a modelof human squamous cell carcinoma. These seeded cultured skin tissue canthen be used to screen compounds or other treatment strategies (e.g.,radiation or tomotherapy) for efficacy against the tumor in its naturalenvironment. Thus, some embodiments of the present invention providemethods comprising providing cultured skin tissue comprising malignantcells or a tumor and at least one test compound, treating the culturedskin tissue with the compound, and assaying the effect of the treatmenton the malignant cells or tumors. In other embodiments of the presentinvention, methods are provided that comprise providing cultured skintissue comprising malignant cells or a tumor and at least one testtherapy (e.g., radiation or phototherapy, treating the cultured skintissue with the therapy, and assaying the effect of the therapy on themalignant cells or tumors.

In other embodiments, cultured skin tissue is used to culture and studyskin pathogens. By way of non-limiting example, cultured skin tissue isinfected with human papilloma virus (HPV) such as HPV18. Methods forpreparing cultured skin tissue infected with HPV are described in U.S.Pat. No. 5,994,115, which is incorporated herein by reference. Thus,some embodiments of the present invention provide methods comprisingproviding cultured skin tissue infected with a pathogen of interest andat least one test compound or treatment and treating the cultured skintissue with the test compound or treatment. In some preferredembodiments, the methods further comprise assaying the effect the testcompound or treatment on the pathogen. Such assays may be conducted byassaying the presence, absence, or quantity of the pathogen in thecultured skin tissue following treatment. For example, an ELISA may beperformed to detect or quantify the pathogen. In some particularlypreferred embodiments, the pathogen is viral pathogen such as HPV.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

In the experimental disclosure which follows, the followingabbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N(Normal); mol (moles); mmol (millimoles); μmmol (micromoles); nmol(nanomoles); g (grams); mg (milligrams); μg (micrograms); ng(nanograms); l or L (liters); ml (milliliters); μl (microliters); cm(centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C.(degrees Centigrade); U (units), mU (milliunits); min. (minutes); sec.(seconds); % (percent); kb (kilobase); by (base pair); PCR (polymerasechain reaction); BSA (bovine serum albumin); Pfu (Pyrococcus furiosus).

Example 1 Keratin 14 Promoter Cloning and Characterization

This Example describes the method used to isolate, clone andcharacterize the K14 promoter DNA. Primer sequences were designed basedon the published K14 Promoter sequence available at Genbank (GenbankAccession #U11076). In order to amplify the 2.35 kb full length K14promoter sequence, the following PCR primers were used:

(SEQ ID NO: 1) Fwd 5′-AAGCTTATATTCCATGCTAGGGTTCTG-3′ (ST080)(SEQ ID NO: 2) Rev 5′-GGTGCAGAGGAGGGAGGTGAGCGA-3′ (ST081)

Human genomic DNA (Promega) was amplified with these primers usingAmplitaq DNA polymerase (Promega). Following a denaturation at 95° C.for 4 minutes, samples were subjected to the following for 30 cycles:denaturation at 95° C. for 1 minute, annealing conditions at 58° C. for1 minute, extension at 72° C. for 3 minutes. A final extension at 72° C.for 7 minutes was followed by a 4° C. hold. The expected PCR product of2.35 kb was observed. This PCR product was gel purified and subsequentlyused for cloning into a TA cloning vector. The pCR 2.1-TOPO TA CloningKit (Invitrogen/LifeTechnologies) was used according to the standardprotocol conditions.

Although thorough sequencing of this promoter has been problematic(typically encountered when sequencing promoter regions presumably dueto the high GC content), the cloned promoter sequence is different thanthe published K14 promoter sequence (Genbank sequence Accession#U110776). The consensus sequence of the cloned Keratin 14 promoterfragment (SEQ ID NO:3) is provided in FIG. 1 a.

In order to confirm the functionality of the K14 promoter sequence, aluciferase reporter gene expression system was used. The K14 promoterfragment was shuttled into the Hind III site of the pGL3 fireflyluciferase vector multiple cloning site. After subcloning this fulllength K14 promoter Hind III fragment an opportunity to truncate thepromoter fragment by approximately 300 bp was easily accomplished usinga single Sma I restriction enzyme site upstream in the multiple cloningsite to release a 300 bp 5′ promoter fragment. Published experimentsdemonstrate a similar 5′ truncation of the K14 promoter reduces thepromoter activity by about 30% (Leask et al., Genes Dev. 4(11):1985-1998(1990)). The full length promoter fragment (2.3 kb) firefly luciferaseactivity was compared to that of the 5′ truncated Promoter fragment(˜2.0 kb) activity. The K14 Promoter Luciferase Vector Construction isdescribed in FIG. 2

Results of luciferase reporter gene expression are as follows. Theco-expression of Renilla Luciferase was used to correct for anyvariability introduced by potentially different transfectionefficiencies or possible differences in cell numbers. Afternormalization, the firefly luciferase reporter gene results demonstratestrong promoter activity from the full length (2.3 kb) K14 promoterfragment and approximately a 30% reduction in firefly luciferaseactivity in the truncated promoter fragment. This result is consistentwith that reported by Leask et al.

Next, the full length K14 promoter was shuttled into the blasticidinselection vector.

Example 2 KGF-2 Cloning and Characterization

This Example describes the isolation, cloning and characterization ofKGF-2. Primer sequences were designed based on the published KGF-2sequence available at Genbank. In order to amplify the 627 bp fulllength KGF-2 sequence, the following PCR primer sequences (BamH I-EcoRV) were used:

(SEQ ID NO: 4) Fwd 5′-CGCGGATCCGCGATGTGGAAATGGATACTG-3′ (ST127)(SEQ ID NO: 5) Rev 5′-GGGATATCCTATGAGTGTACCACCATTGGA-3′ (ST128)

Pfu Turbo DNA Polymerase (Stratagene) was used to minimize the risk ofPCR induced errors. Human Universal QUICK-Clone cDNA (CLONTECH) was usedas the template for PCR amplification of the full length KGF-2 cDNA.Following a denaturation at 94° C. for 4 minutes, samples were subjectedto the following for 30 cycles: denaturation at 94° C. for 30 seconds,annealing conditions at 51° C. for 30 seconds, extension at 72° C. for 1minute. A final extension at 72° C. for 7 minutes was followed by a 4°C. Hold. The expected PCR product of 627 bp was observed. Afteramplification, the addition of 3′ A-overhangs to the Pfu PCR product wasnecessary to allow for efficient TA cloning. The PCR product was gelpurified using a Matrix Gel Extraction System (Marligen BioScienceInc.). Gel purified PCR products were cloned into a commerciallyavailable TA cloning kit. The pCR 2.1-TOPO TA Cloning Kit(Invitrogen/Lifetechnologies) was used according to the standardprotocol conditions.

Sequencing reactions were performed using each of the two sequencingprimers that span the cloning site (M13 forward and reverse primers).Additionally, overlapping sequence was obtained using the KGF-2 specificprimers used to PCR amplify the cDNA (Primers ST127 and ST128). The cDNAsequence was identical to Genbank accession #U67918.

Next, the TA cloned KGF-2 cDNA was shuttled into a pIRES vector. The TAclone containing the correct KGF-2 cDNA sequence was digested with BamHI(5′) and EcoRV (3′) to release a 627 bp KGF-2 cDNA. This product wascloned directly into the BamHI and EcoRV sites of the mammalianexpression pIRESpuro clonal selection vector.

A re-amplification and TA cloning step was necessary to obtain thedesired restriction enzyme sites for directional cloning into thepUB-Bsd clonal selection vector. The primer sequences used to amplifythe KGF-2 cDNA that contain the Not I and Sal I restriction enzyme sitesare as follows.

(SEQ ID NO: 109) Fwd 5′-GCGGCCGCATGTGGAAATGGATACTG-3′ (ST133)(SEQ ID NO: 110) Rev 5′-GTCGACCTATGAGTGTACCACCATTGGA-3′ (ST134)

The PCR conditions were the same as listed above. Pfu polymerase(Stratagene) was used, but fewer PCR cycles were required because theprevious TA clone containing the KGF-2 gene was used as the startingtemplate for this additional round of amplification.

The PCR product contained strategically placed Not I and Sal Irestriction enzyme sites. This PCR product was cloned with the pCR2.1-TOPO TA Cloning Kit (Invitrogen/Lifetechnologies) according to thestandard protocol conditions.

The newly cloned KGF-2 cDNA was sequenced, and the sequence wasconfirmed to be identical to the KGF-2 cDNA sequence (Genbank accession#U67918).

The KGF-2 cDNA clone was shuttled out of the TA cloning vector bydigestion with a 5′ Not I and a 3′ Sal I (ligates to Xho I restrictionenzyme cleavage site) restriction enzyme. This fragment wasdirectionally cloned between the K14 promoter and the globin polyAsequences in the pUB-Bsd vector using the Not I and Xho I restrictionenzyme sites.

Example 3 Mammalian Expression Vector Design

This Example presents a mammalian expression vector utilized in thepresent invention. The vector is described in FIG. 3 and comprises thefollowing elements: K14 promoter (2.35 kb)/KGF-2 cDNA (627 bp)/globinintron & poly(A) (1.165 kb)/pUB-Bsd (4.245 kb).

Example 4 KGF-2 mRNA Expression Diagnostic Screen (RT-PCR)

This Example describes the KGF-2 mRNA expression diagnostic screenutilized in the present invention. NIKS cells were transfected usingTrans-It Keratinocyte Transfection Reagent (Mirus Corp.) and grown ineither EpiLife Medium (Cascade Biologics) or NIKS STRATALIFE medium(Stratatech Corporation). Supernatants were collected for three days andused in the development of a direct KGF-2 ELISA Assay. After three daysthe cells were lysed with Trizol Reagent (Invitrogen) for RNA isolation.First strand cDNA synthesis was performed using total RNA isolated formthese transiently transfected NIKS cells. The following primer sequenceswere utilized:

(SEQ ID NO: 6) Fwd 5′-TGCTGTTCTTGGTGTCTTCCG-3′ (ST135) (SEQ ID NO: 7)KGF-2 Specific Rev 5′-CAACCAGCACGTTGCCCAGG-3′ (ST124) (SEQ ID NO: 8)Globin fragment Specific Oligo d(T) 5′TGTTACCAATCTGAAGTGGGAGCGGCCGCCCTTTTTTTTTTTTTTTTTTT-3′ (ST112)

Next, reverse transcriptase reactions were conducted under the followingconditions: RNA Priming Reaction-2.5 ug total RNA (Template), 0.5 mMdNTP mix, Oligo dT (0.5 ug)—Incubate 65 for 5 minutes, on ice 3 minutes.First strand cDNA synthesis reaction (added to the RNA PrimingReaction)-1×RT buffer (Promega Corp.), Rnase Out (40 U) (Invitrogen),M-MLV RT (200U) (Promega), 42 degrees for 50 minutes, heat 70 degreesfor 15 minutes. One microliter (1 ul) of RT reaction template was usedfor the subsequent PCR reaction.

Next, PCR was conducted. Following a denaturation at 95° C. for 5minutes, samples were subjected to the following for 35 cycles:Denaturation at 94° C. for 30 seconds, Annealing conditions at 60° C.for 30 seconds, Extension at 72° C. for 1 minute. A final Extension at72° C. for 7 minutes was followed by a 4° C. Hold. The RT-PCR strategyis diagrammed in FIG. 4.

A DNA vector specific product of 1.1 kb was observed along with thespecific product associated with first strand cDNA synthesis (KGF-2 RNAspecific Product) of approximately 600 bp was observed. No KGF-2 RNAspecific product was observed in either the mock (vector w/o KGF-2 cDNAinsert) control plasmid transfection or the reverse transcriptase minuscontrol reaction.

Example 5 KGF-2 Protein Expression Diagnostic Screen (Direct ELISA)

This Example describes the KGF-2 protein expression diagnostic screenused in the present invention.

NIKS cells were transfected using Trans-It Keratinocyte TransfectionReagent (Mirus Corp.) and grown in either EpiLife Medium (CascadeBiologics) or NIKS medium (Stratatech Corporation). Supernatants werecollected for three days and used in the development of a direct KGF-2ELISA Assay. The 100 ul supernatants were incubated in plate (NuncImmunoassay plate) over night; at a minimum samples were plated induplicate. The next day, the samples were washed 3× (1×PBS/0.05%Tween-20) 300 ul/well; blocked plate (1×PBS/1% BSA/5% Sucrose) 300ul/well @rt for 30 minutes; washed 3×(1×PBS/0.05% Tween-20) 300 ul/well;incubated with rabbit anti-huKGF-2 Ab (0.2 ug/well) @rt for 2 hours;washed 3×(1×PBS/0.05% Tween-20) 300 ul/well; incubated with goatanti-rabbit HRP (0.8 mg/ml) Ab—use at 1:1000 dilution @rt for 30minutes; washed 3×(1×PBS/0.05% Tween-20) 300 ul/well; prewarmed TMB @rt100 ul/well for 30 minutes at room temperature; added 50 ul of 2N H2SO4;read O.D. 450 nm and 620 nm; corrected for plate imperfections (450nm-620 nm).

This experiment demonstrates elevated KGF-2 protein levels are detectedin the supernatants of transiently transfected NIKS cells, when comparedto either mock transfection (empty vector) or medium alone controls.

Example 6 Isolation of NIKS Cells Expressing Exogenously Introduced FullLength Human KGF-2 Protein

This Example describes the isolation of NIKS cells that express KGF-2.

A. Clonal Isolation Strategy—

Vector Construct—Keratin 14 promoter/KGF-2 cDNA/pUb-Bsd plasmid.

A DNA fragment encoding KGF-2 was isolated by PCR and sequenced toverify the identity and integrity of the PCR product. The DNA fragmentwas identical to previously reported sequences for KGF-2. The DNAfragment encoding KGF-2 was cloned into a mammalian expression vectorcontaining a blasticidin resistant cassette. Blasticidin has been usedto select for stably transfected keratinocytes, which are subsequentlyable to undergo normal differentiation.

To provide for constitutive expression of KGF-2 in keratinocytes of thebasal epidermal layer, constructs were generated in which expression ofKGF-2 is under the control of the human keratin-14 (K14) promoter. A 2.3kb genomic DNA fragment containing the K14 promoter was amplified andits activity was confirmed by the ability to promote luciferaseexpression from the pGL3 reporter plasmid (Promega) in NIKS cells. The2.3 kb K14 promoter was then cloned into the pUb-bsd vector(Invitrogen). Subsequently, the KGF-2 coding region was cloneddownstream of the K14 promoter and a DNA fragment containing the rabbitβ-globin intron and poly (A) signal was inserted downstream of the KGF-2coding region to complete this mammalian expression vector construction.

The structure of the final vector was confirmed by restriction enzymemapping and DNA sequencing. Oligonucleotide primers were synthesized andused to examine the expression of this construct in NIKS keratinocytecells using semi-quantitative RT-PCR analysis. The primers were designedto span an intron in the rabbit β-globin fragment, such that PCRproducts generated from a spliced RNA template is approximately 500 bpsmaller than the corresponding fragment amplified from genomic DNA.

Transfection—

Transit-Keratinocyte (Minis) transfection reagent was used to introducethe KGF-2 vector DNA into monolayer NIKS cell cultures. Twenty-four toforty-eight hours post transfection the NIKS cells were plated onto ablasticidin feeder layer of cells and fed with blasticidin selectionmedium.

Selection—

NIKS keratinocyte clones were cocultured in the presence of blasticidinresistance feeder cells and selected for growth in presence of NIKS™medium containing 2.5 ug/ml blasticidin. Only those colonies thatcontinued to grow in the presence of blasticidin selection for durationof selection (a minimum of 18 days) were isolated and expanded forfurther characterization.

Clone Isolation—

A traditional “Ring cloning” method to isolate blasticidin resistantcolonies re-plated to individual tissue culture plates (p35 and p100)containing mouse fibroblast feeder cells. When these cultures reach80-90% confluence, the p35 cultures are harvested for expressionanalysis and the p100 cultures are used for the subsequent expansionphase.

Characterization of Stably-transfected NIKS keratinocytes—

Stable NIKS keratinocyte colonies that survived the selection schemetherefore are presumed to contain the K14-KGF-2 expression construct. Toconfirm the presence of the KGF-2 transgene, genomic DNA was isolatedfrom each clone and amplified with vector specific primers. This PCRscreen was designed to reconcile products derived from transgene DNAfrom that of potential endogenous KGF-2 DNA products. Multiple cloneswere obtained using this construct and associated selection scheme.

Expansion—

The results of expression analysis obtained from the p35 culturesdictate which clones will be expanded for further characterization. Thep100 plates from cultures identified as having positive expression aregrown to approximately 50-80% confluence then expanded onto severalplates containing mouse fibroblast feeder cells.

B. Results

Twenty-nine NIKS clonal isolates that survived drug selection wereisolated and characterized. Four of the 29 originally identified clonesdid not survive the expansion phase. The remaining 25 clones weresuccessfully expanded and confirmed to express KGF-2, at the level oftranscription, determined using RT-PCR. Total RNA isolated from previoustransient transfections served as positive RT-PCR controls. Negativecontrols were identical reactions run in the absence of reversetranscriptase. The presence of a KGF-2 transgene present in the genomeof any clone yielded an anticipated PCR product of approximately 1 Kb insize with the use of a transgene specific primer set. Clones werecategorized by semi-quantitative expression analysis into categoriesrepresenting low, medium or high expression levels.

Example 7 KGF-2 RNA and Protein Expression in Monolayer Cultures

This example describes experiments analyzing the expression of KGF-2 inmonolayer cell cultures. Each of the confirmed RT-PCR positive cloneswere assayed for protein expression; this effort resulted in thedetection of KGF-2 protein over expression in supernatants. Commerciallyavailable KGF-2 specific antibodies were used to investigate proteinlevels of secreted KGF-2 protein detected in supernatants. Western Blotand ELISA analysis was performed on cell culture supernatants of clonesand compared to native NIKS cell supernatants. A cell growth assay isbeing developed to investigate possible biological effects ofconditioned media from cultured NIKS KGF-2 clones compared to endogenousNIKS cell supernatants.

A. RT-PCR

Transgene specific PCR products were semi-quantitatively reportedrelative to GAPDH specific products. The transgene specific PCR primerset was designed to produce a product utilizing the rabbit β-globinintron sequence region restricted to the transgene; as a result thisproduct is easily distinguishable from endogenous KGF-2 product.

Transfected cultures were assayed for mRNA expression levelsapproximately 24 hours post-transfection. A commercially available RNAisolation kit was used to isolate total cellular RNA (Invitrogen,Carlsbad, Calif.). Total RNA provided a suitable template for thesubsequent first strand cDNA synthesis (reverse-transcriptase) reactionfollowed by the polymerase chain reaction (RT-PCR). Amplificationproducts are resolved on an ethidium bromide stained agarose gel. Theanticipated PCR products specific for the transgene DNA and mRNA productis 1.0 Kb and 550 bp respectively.

An additional RT-PCR primer set was designed to specifically amplify theKGF-2 gene mRNA product, however this primer set does not distinguishbetween endogenous and transgene messages. Despite the inability todistinguish endogenous mRNA from transgene mRNA intensities weresemi-quantitatively compared using the endogenous control samples(untransfected and transfected with empty vector) as a point ofreference.

To compare the level of KGF-2 RNA expressed from the K14-KGF-2 constructwith KGF-2 RNA from the endogenous gene, RT-PCR analysis was performedusing primers that will amplify KGF-2 RNA regardless of its origin.Under these conditions endogenous KGF-2 has not been identified usingthese RT-PCR conditions, therefore, KGF-2 does not appear to beexpressed in NIKS keratinocytes. To date, no KGF-2 RT-PCR products fromnon-transfected NIKS cell total RNA controls have been identified. Theanticipated 550 bp fragment is routinely observed in NIKS cellstransfected with the KGF-2 transgene. The KGF-2 expressed from theK14-KGF-2 construct gives rise to the 550 bp RT-PCR product. RT-PCRanalysis of two K14-KGF-2 clones show that the 550 bp KGF-2 RNA productis overexpressed compared to non-detected endogenous KGF-2 levels. NoPCR products were seen in control reactions in which reversetranscriptase was omitted, demonstrating that these products are derivedfrom RNA and not from template contamination of the PCR reactions. Theseresults demonstrate that NIKS clones stably-transfected with theK14-KGF-2 expression construct specifically overexpress the KGF-2transgene.

B. Western Blot

Western blot analysis demonstrates specific products at anticipated gelpositions that correspond to post translational modification forms ofKGF-2 reported in the literature. Prominent KGF-2 specific protein bandsare observed between 19 and 30 kDa. Specific KGF-2 band productintensities observed in Western blot analysis corroborate thesemi-quantitative RT-PCR expression results. Endogenous KGF-2 is notdetected in unmodified NIKS control cultures; these findings areconsistent with results obtained from semi-quantitative mRNA expressionanalysis. A positive control (recombinant human KGF-2) protein was usedat concentrations ranging from 0.3 to 0.5 ng/lane that routinelycorresponds with the 19 kDa KGF-2 protein band.

To quantify KGF-2 protein expression in stably-transfected K14-KGF-2clones, a KGF-2 Sandwich ELISA (Polyclonal antibodies from R&D Systemsand Santa Cruz) was developed to compare KGF-2 levels between variousK14-KGF-2 clones and untransfected NIKS cells. Supernatants from severalK14-KGF-2 clones contain elevated levels of KGF-2 compared to unmodifiedNIKS cell control samples. This increase in KGF-2 protein expression isconsistent with the increase seen by RT-PCR analysis. These resultsdemonstrate that NIKS cells can be engineered to stably express andsecrete elevated levels of KGF-2 protein.

C. ELISA

A Sandwich assay was developed to compare secreted KGF-2 levels; assayresults are reported as amount of protein detected per milliliter ofcell supernatant. The level of KGF-2 protein detected in supernatants iswell above levels detected in unmodified NIKS cell supernatants(negative control) samples. ELISA values were obtained for individualclones and used to assign relative expression levels.

Taken together, the expression analysis compiled from each of theseassays was used to group clones into relative expression levels whencompared to one another.

Example 8 KGF-2 RNA and Protein Expression in Organotypic Cultures

This example describes experiments analyzing the expression of KGF-2 inorganotypic cultures.

A. RT-PCR—Comparison of Biopsy Samples (Clones Versus NIKS)

The expression of KGF-2 mRNA was examined by RT-PCR in skin tissuegenerated from stable clones. Total RNA was extracted from skin tissueand subjected to RT-PCR using primers that detect mRNA expressed fromthe KGF-2 transgene, but not from an endogenous KGF-2 gene. KGF-2 mRNAwas detected in skin tissue prepared from a K14-KGF-2 clone, but was notdetected in RNA from skin tissue prepared from untransfected NIKS cells.These results demonstrate that the K14-KGF-2 construct is expressedwithin the context of stratified epidermis.

B. Western Blot

Results were similar to those obtained for the monolayer cell cultures.

C. ELISA

Results were similar to those obtained for the monolayer cell cultures.

D. Histology—Biopsy of Clones Versus NIKS

To verify that stably-transfected clones containing the K14-KGF-2expression constructs undergo normal epidermal differentiation, culturedskin tissue containing these clones was prepared. After two weeks inorganotypic culture, K14-KGF-2 clones formed cultured skin tissue withnormal epidermal morphology. These findings indicate that elevatedexpression of KGF-2 does not interfere with the ability of NIKS cells toundergo normal epidermal differentiation.

Example 9 Use of Skin Equivalents Expressing Exogenous KGF-2 to CloseWounds

This Example describes preliminary experimental results obtained whenskin equivalents expressing exogenous KGF-2 were used to close wounds ina mouse wound model. In this experiment, organotypic cultured skin(i.e., skin equivalents) were grafted onto the denuded back of athymicnude mice. Skin equivalents containing native NIKS cells were comparedto genetically modified skin equivalents expressing KGF-2. All tissueswere meshed (2:1 ratio) immediately prior to being grafted onto mice.Interstitial wound space closure was monitored in the mice. Eachobservation time point included recording micrometer measurements of thewound area; these measurements were supplemented with digitalphotography. At post operative day 3 (POD 3), complete wound closure ofinterstitial spaces have been observed in the mice with the geneticallymodified NIKS organotypic skin tissue (KGF-2), but not observed in micegrafted with the NIKS culture tissue control.

Example 10 Mammalian Expression Vector Design

This Example describes a mammalian expression vector utilized in someembodiments of the present invention. The vector is described in FIG. 5and comprises the following elements: Involucrin promoter (3.7 kb)/KGF-2cDNA (627 bp)/globin intron & poly(A) (1.165 kb)/pUB-Bsd (4.245 kb).

Construction of Expression Vector

A genomic DNA fragment containing the human involucrin promoter sequencewas isolated using PCR primers based on published sequences (Crish etal., J Biol Chem, 1998. 273(46): p. 30460-5). The integrity of thecloned involucrin promoter PCR product was confirmed by restrictionenzyme analysis and DNA sequencing using involucrin specific primers.The involucrin promoter is not expressed in undifferentiatedkeratinocytes, but is specifically activated in differentiatedkeratinocytes. It is preferable to direct overexpression of the KGF-2 todifferentiated keratinocytes to avoid interfering with normalkeratinocyte differentiation.

The coding region for the KGF-2 gene is cloned into the pUB-Bsdexpression vector (Invitrogen, Carlsbad, Calif.). This vector ismodified by inserting the involucrin promoter upstream of the multiplecloning site. This vector contains the blasticidin drug selectioncassette that utilizes the ubiquitin promoter sequence drivingblasticidin gene expression. Briefly, gene specific primers for KGF-2were designed to contain terminal restriction enzyme sites (5′-Eco RVand 3′-Spe I). These primers were used in a PCR reaction containing TAcloned cDNA template. The modified KGF-2 PCR product (containingterminal restriction enzyme sites) was cloned into the TA cloning vector(Invitrogen) then sequenced. The KGF-2 cDNA gene product was shuttledfrom the TA cloning vector into a mammalian expression vector. Completemammalian expression vector construction required a two step vectorassembly approach shown in FIG. 5.

KGF-2 mRNA Expression Diagnostic Screen (RT-PCR)

A mRNA expression screen was performed as described in Example 4.

Involucrin Promoter/KGF-2 Expression Construct

1) Electroporation Transfection Method Results—

TABLE 1 Summary of clonal selection and mRNA expression results.Experiment Clones Picked Clones Survived Positive 54:29 4 4 4 54:31 2 22 68:31 5 3 22) Trans-IT Keratinocyte Transfection Method Results—

TABLE 2 Summary of clonal selection and mRNA expression results.Experiment Clones Picked Clones Survived Positive 58:51 (TransIT) 16 2 2

Isolation of NIKS Cells Expressing Exogenously Introduced Full LengthHuman KGF-2 Protein

A. Clonal Isolation Strategy—

Vector Construct—

This clonal isolation strategy includes the use of a DNA maxiprep(Qiagen) of the Involucrin/KGF-2 cDNA/Globin poly(A) fragment/pUb-Bsdplasmid.

TransIT-keratinocyte Transfection Method—

Transit-Keratinocyte (Minis) transfection reagent was used to introducethe KGF-2 vector DNA into monolayer NIKS cell cultures. Twenty-four toforty-eight hours post transfection the NIKS cells were plated onto ablasticidin feeder layer of cells and fed with blasticidin selectionmedium.

Electroporation Transfection Method—

Early passage NIKS cells were harvested at @approximately 50-70%confluence. Cells were pelleted and the pellet resuspended (2×10⁶cells/800 ul) in F-12/DME (5:1).

800 ul of NIKS cell suspension was placed in 0.4 cm electroporationcuvette, DNA was added (10-30 ug, linear or supercoiled), placed incuvette holder of the GenePulser and started. All steps were done atroom temperature; the cells were not placed on ice at any time duringthis procedure. The actual voltage and capacitance values were recorded

Electroporated NIKS cells were removed from the cuvette and diluted into25-50 mls of fresh NIKS medium, mixed well by pipetting, and plated(5-10 mls) per p150 containing blasticidin resistant feeders (usingeither 5 or 10 p150's per transfection reaction).

The following day, the medium is replaced on the p150's with blasticidincontaining medium (2.5 ug/ml blasticidin).

BioRad GenePulser Electroporation Settings:

Exponential Pulse Program

270 volts

950 uF

∞ ohms

0.4 cm cuvette

Selection—

NIKS keratinocyte clones were cocultured in the presence of blasticidinresistance feeder cells and selected for growth in presence of NIKSmedium containing 2.5 ug/ml blasticidin. Only those colonies thatcontinued to grow in the presence of blasticidin selection for durationof selection (a minimum of 18 days) were isolated and expanded forfurther characterization.

Clone Isolation—

A traditional “Ring cloning” method to isolate blasticidin resistantcolonies re-plated to individual tissue culture plates (p35 and p100)containing mouse fibroblast feeder cells. When these cultures reach80-90% confluence, the p35 cultures are harvested for expressionanalysis and the p100 cultures are used for the subsequent expansionphase.

Characterization of Stably-transfected NIKS keratinocytes—

Stable NIKS keratinocyte colonies that survived the selection schemetherefore are presumed to contain the Involucrin-KGF-2 expressionconstruct. To confirm expression of the KGF-2 transgene, total RNA wasisolated from each clone to provide a template for RT-PCR analysis.Multiple clones were obtained using this construct and associatedselection scheme.

Expansion—

The results of expression analysis obtained from the p35 culturesdictate which clones will be expanded for further characterization. Thep100 plates from cultures identified as having positive expression aregrown to approximately 50-80% confluence and then expanded onto severalplates containing mouse fibroblast feeder cells.

B. Results—

TransIT-Kerationcyte Method of Transfection for Clonal Selection—

Sixteen NIKS clonal isolates that survived drug selection were isolatedand characterized. Only two of the 16 originally identified clonessurvived the expansion phase. These two clones were successfullyexpanded and confirmed to express KGF-2, at the level of transcription,determined using RT-PCR. Total RNA isolated from previous transienttransfections served as positive RT-PCR controls. Negative controls wereidentical reactions run in the absence of reverse transcriptase. Thepresence of a KGF-2 transgene present in the genome of any clone yieldedan anticipated PCR product of approximately 1 Kb in size with the use ofa transgene specific primer set. Clones were categorized bysemi-quantitative expression analysis into categories representing low,medium or high expression levels.

Electroporation Method Transfection for Clonal Selection—

In one selection experiment, Four NIKS clonal isolates that surviveddrug selection were isolated and characterized. All four originallyidentified clones survived the expansion phase. In a second experiment,Two NIKS clonal isolates that survived drug selection were isolated andcharacterized. Both originally identified clones survived the expansionphase. In a third experiment, Five NIKS clonal isolates that surviveddrug selection were isolated and characterized. All five originallyidentified clones survived the expansion phase.

All clones generated in this series of selection experiments weresuccessfully expanded and confirmed to express KGF-2, at the level oftranscription, determined using RT-PCR. Total RNA isolated from previoustransient transfections served as positive RT-PCR controls. Negativecontrols were identical reactions run in the absence of reversetranscriptase. The presence of a KGF-2 transgene present in the genomeof any clone yielded an anticipated PCR product of approximately 1 Kb insize with the use of a transgene specific primer set. Clones werecategorized by semi-quantitative expression analysis into categoriesrepresenting low, medium or high expression levels.

Example 11 Expression of Endogenous Human Beta Defensins in NIKS Cells

This example provides an analysis of endogenous human beta defensin(hBD) expression in NIKS cells. Since it was unknown if NIKS cellsexpress hBDs, RT-PCR analysis was performed to verify detectable levelsof hBD-1, hBD-2, and hBD-3 in both monolayer and organotypic cultures ofNIKS keratinocytes. Specifically, reverse transcriptase reactions wereperformed on both monolayer and organotypic NIKS cell cultures for eachof the human β-defensin genes being studied. Reverse transcriptasereactions were performed using total RNA isolated from both NIKS cellmonolayer and organotypic cultures using an oligonucleotide d(T) primer.One microliter of RT reaction template was used in a 20 ul PCR reactioncontaining gene specific primers. PCR reactions were conducted asfollows—Denaturation at 95° C. for 5 minutes, samples were subjected tothe following for 35 cycles: Denaturation at 94° C. for 30 seconds,Annealing conditions at 58° C. for 30 seconds, Extension at 72° C. for30 seconds. A final Extension at 72° C. for 7 minutes was followed by a4° C. Hold. Fifteen microliters of a 20 ul PCR reaction was resolved ona 1% agarose gel containing ethidium bromide. The gels were analyzed forthe anticipated PCR product sizes of 275 bp, 205 bp & 290 bpcorresponding to hBD-1, hBD-2 & hBD-3 respectively.

Intact human skin is reported to express all three human hBDs and theirexpression levels are increased in response to injury and inflammation.To date there have been no reports on the expression of hBDs in primaryhuman keratinocytes in monolayer and only one report on hBD-2 proteinexpression in a nontherapeutic product, Matek's EpiDerm. A thoroughanalysis of the RNA expression levels of all three hBDs in bothmonolayer and organotypic cultures of NIKS keratinocytes was conducted.Organotypic culture of NIKS keratinocytes results in enhanced levels ofall hBDs relative to monolayer culture conditions, although themagnitude of induction varied among the hBDs. In monolayers of NIKScells the steady state mRNA expression levels of hBD-2 and hBD-3 werebelow the limit of detection. hBD-3, a broad spectrum antimicrobialpeptide, was poorly expressed even in organotypic culture supporting thenotion that overexpression of hBD-3 in NIKS keratinocytes will result inenhanced antimicrobial properties especially in bioengineered human skintissue generated by organotypic culture techniques.

Example 12 Cloning of Human Beta Defensin

This example describes the cloning of hBD-1, h-BD2, and hBD3 from NIKScells. The reverse transcriptase-polymerase chain reaction productsdescribed in Example 1 were cloned into the TA cloning vector(Invitrogen) and sequenced to confirm their genetic identity. A summaryof the sequencing results for each of the cloned cDNA products is asfollows. Human β-defensin-1 cDNA sequence was confirmed to be identicalto Genbank Accession #U73945 for hBD-1. Sequence of the humanβ-defensin-2 cDNA reveled a point mutation at amino acid position #48(Lys→Arg) when compared to Genbank Accession #AF040153 for hBD-2. Thesequence was amplified, using Pfu proof-reading polymerase, and cloned.The sequence was confirmed to be identical to the GenBank sequence.

The sequence of the human β-defensin-3 cDNA clone was originally foundto contain two point mutations at amino acid positions #57 (Thr→Met) and#62(Cys→Tyr). Pfu polymerase (proof-reading enzyme) was used tosuccessfully re-amplify the hBD-3 cDNA which was cloned into the TAcloning vector and sequenced. The sequence of this new clone isidentical to that reported in the Genbank Accession #AF295370 for hBD-3.

Example 13 Construction of Expression Vectors

The example describes the construction of hBD expression vectors. Agenomic DNA fragment containing the human involucrin promoter sequencewas isolated using PCR primers based on published sequences. Crish, J.F., T. M. Zaim, and R. L. Eckert, The distal regulatory region of thehuman involucrin promoter is required for expression in epidermis. JBiol Chem, 1998. 273(46): p. 30460-5. The integrity of the clonedinvolucrin promoter PCR product was confirmed by restriction enzymeanalysis and DNA sequencing using involucrin specific primers. Theinvolucrin promoter is not expressed in undifferentiated keratinocytes,but is specifically activated in differentiated keratinocytes. Inprevious studies, we have demonstrated the use of this involucrinpromoter fragment support expression in monolayer cultures of NIKSkeratinocytes. It is preferable to direct overexpression of theβ-defensins to differentiated keratinocytes to avoid interfering withnormal keratinocyte differentiation.

The coding region for each of the β-defensin genes is cloned into thepUB-Bsd expression vector (Invitrogen, Carlsbad, Calif.). This vector ismodified by inserting the involucrin promoter upstream of the multiplecloning site. This vector contains the blasticidin drug selectioncassette that utilizes the ubiquitin promoter sequence drivingblasticidin gene expression. A restriction enzyme map of the hBD1 vectoris provided in FIG. 12. Briefly, gene specific primers for hBD-1 weredesigned to contain terminal restriction enzyme sites (5′-Xma 1 and3′-Xba I). These primers were used in an RT-PCR reaction containingtotal cellular RNA isolated from NIKS cells. The hBD-1 PCR product wascloned into the TA cloning vector (Invitrogen) then sequenced. Thedefensin cDNA gene product was shuttled from the TA cloning vector intoa mammalian expression vector. Complete mammalian expression vectorconstruction required a two step vector assembly approach shown in FIG.13. A similar cloning strategy was used to generate the hBD-2 and hBD-3mammalian expression constructs.

Example 14 Expression of Exogenous hBD in NIKS Cells

Purified DNA from each of the Involucrin-β-defensin-UB-Bsd vectors wasintroduced into NIKS cells. Specifically, NIKS cells were transfectedusing Translt-Keratinocyte reagent (Mirus Corporation), which has beenused to efficiently transfect NIKS cells. Negative control samplesincluded mock transfected (no DNA) or empty vector (no β-defensin)transfected populations of NIKS cells.

mRNA analysis:

Transfected cultures were assayed for mRNA expression levelsapproximately 24 hrs post-transfection. A commercially available RNAisolation kit was used to isolate total cellular RNA (Invitrogen,Carlsbad, Calif.). Total RNA provided a suitable template for thesubsequent first strand cDNA synthesis (reverse-transcriptase) reactionfollowed by the polymerase chain reaction (RT-PCR). Amplificationproducts were resolved on an ethidium bromide stained agarose gel. Theanticipated PCR products specific for the transgene DNA and mRNA is asfollows—hBD-1 (720 bp and 220 bp), hBD-2 (700 bp and 200 bp), and hBD-3(710 bp and 210 bp) respectively.

Results of this experiment confirm the anticipated RT-PCR product sizes,for each of the three defensins. Also, as anticipated in the reversetranscriptase minus control reactions a single robust signal wasdetected which corresponds to the amplification of the transgene DNA. Nospecific PCR products were observed in the mock transfected controlreactions.

Protein Analysis:

Culture medium from cells transiently transfected with each of the threecandidate β-defensin transgenes is assayed for β-defensin peptideproduction using an Enzyme-Linked Immunosorbent Assay (ELISA) andWestern Blotting assays using anti-β-defensin antibodies (Santa CruzBiotechnology, Santa Cruz, Calif.). A comparison is made to endogenouslevels from non-transfected NIKS cells. A synthetic peptide, positivecontrol, is included in these assays. Cell lysates may be required foranalysis as β-defensin protein may remain associated with the outermembranes of the cells rather than freely secreted into the culturemedium.

Example 15 Isolation of NIKS Cells Expressing Exogenously IntroducedFull Length hBD-1 Protein

This Example describes the isolation of NIKS cells that express hBD-1.

A. Clonal Isolation Strategy

Vector Construct—Involucrin promoter/hBD-1 cDNA/pUb-Bsd Plasmid.

A DNA fragment encoding hBD-1 was isolated by PCR and sequenced toverify the identity and integrity of the PCR product. The DNA fragmentwas identical to previously reported sequences for hBD-1. The DNAfragment encoding hBD-1 was cloned into a mammalian expression vectorcontaining a blasticidin resistant cassette. Blasticidin has been usedto select for stably transfected keratinocytes, which are subsequentlyable to undergo normal differentiation.

To provide for constitutive expression of hBD-1 in keratinocytes of thestratified epidermal layer, constructs were generated in whichexpression of hBD-1 is under the control of the human Involucrinpromoter. A 3.7 kb genomic DNA fragment containing the Involucrinpromoter was amplified then cloned into the pUb-bsd vector (Invitrogen).The hBD-1 coding region was cloned downstream of the Involucrin promoterand a DNA fragment containing the rabbit β-globin intron and poly (A)signal was inserted downstream of the hBD-1 coding region to completethis mammalian expression vector construction.

The structure of the final vector was confirmed by restriction enzymemapping and DNA sequencing. Oligonucleotide primers were synthesized andused to examine the expression of this construct in NIKS keratinocytecells using semi-quantitative RT-PCR analysis. The primers were designedto span an intron in the rabbit β-globin fragment, such that PCRproducts generated from a spliced RNA template are approximately 500 bpsmaller than the corresponding fragment amplified from genomic DNA.

Transfection—

Transit-Keratinocyte (Minis) transfection reagent was used to introducethe hBD-1 vector DNA into monolayer NIKS cell cultures. Twenty-four toforty-eight hours post transfection the NIKS cells were plated onto ablasticidin feeder layer of cells and fed with blasticidin selectionmedium.

Selection—

NIKS keratinocyte clones were cocultured in the presence of blasticidinresistance feeder cells and selected for growth in presence of NIKSmedium containing 2.5 ug/ml blasticidin. Only those colonies thatcontinued to grow in the presence of blasticidin selection for durationof selection (a minimum of 18 days) were isolated and expanded forfurther characterization.

Clone Isolation—

A traditional “Ring cloning” method was used to isolate blasticidinresistant colonies re-plated to individual tissue culture plates (p35and p100) containing mouse fibroblast feeder cells. When these culturesreach 80-90% confluence, the p35 cultures are harvested for expressionanalysis and the p100 cultures are used for the subsequent expansionphase.

Characterization of Stably-transfected NIKS Keratinocytes—

Stable NIKS keratinocyte colonies that survived the selection schemetherefore are presumed to contain the Involucrin-hBD-1 expressionconstruct. To confirm the presence of the hBD-1 transgene, total RNA wasisolated from each clone and RT-PCR amplified with transgene specificprimers. This PCR screen was designed to reconcile products derived fromtransgene total RNA from that of potential endogenous hBD-1 expressionproducts. Multiple clones were obtained using this construct andassociated selection scheme.

Expansion—

The results of expression analysis obtained from the p35 culturesdictate which clones were expanded for further characterization. Thep100 plates from cultures identified as having positive expression weregrown to approximately 50-80% confluence then expanded onto severalplates containing mouse fibroblast feeder cells.

B. Results

Thirty NIKS clonal isolates that survived drug selection were isolatedand characterized. Ten of the 30 originally identified clones did notsurvive the expansion phase. The remaining 20 clones were successfullyexpanded and confirmed to express hBD-1, at the level of transcription,determined using RT-PCR. Total RNA isolated from previous transienttransfections served as positive RT-PCR controls. Negative controls wereidentical reactions run in the absence of reverse transcriptase. Thepresence of a hBD-1 transgene detected in the genome of any cloneyielded an anticipated PCR product of approximately 720 bp in size withthe use of a transgene specific primer set. Clones were categorized bysemi-quantitative expression analysis into categories representing low,medium or high expression levels.

NIKS cell expression of exogenously introduced full length hBD-3 proteinhave also been isolated in the same fashion as described above.

Example 16 hBD Activity in NIKS Cells

This Example describes assays for hBD activity. To determine iftransient expression of β-defensins in NIKS monolayer cultures resultsin enhanced bactericidal activity, a modified in vitro inhibition zoneassay is utilized. Hultmark, D., et al., Insect immunity. Attacins, afamily of antibacterial proteins from Hyalophora cecropia. Embo J, 1983.2(4): p. 571-6. Briefly, thin (1 mm) agarose plates are seeded with amicrobe of choice (E. coli, S. aureus, P. aeruginosa, S. pyogenes or C.albicans). The melted agarose (1%) contains Luria-Bertani broth with orwithout supplemented salt. Vogel, H. J., Acetylornithinase ofEscherichia coli: partial purification and some properties. J Biol Chem,1956. 218: p. 97-106. The test organism, (˜5×10⁴ log-phase cells/ml) isadded just before pouring the plate. Small wells (3 mm diameter) arepunched in the assay plates and loaded with 3 ul of harvested culturemedium conditioned for at least 24 hours by untransfected NIKS, NIKStransiently transfected with the empty expression construct, or NIKStransiently expressing each β-defensin. Alternatively, discs are loadedwith 3 ul of harvested conditioned medium described above and placed ona plate containing the seeded microbial lawn. A positive control sampleof a synthetic hBD-3 peptide (2-30 ug/ml) or an antibiotic such asstreptomycin (100 ug/ml) is added to the conditioned medium and assayed,along with a negative control (unconditioned medium sample). Afterovernight incubation at 30° C., the inhibition zones are recorded usinga ruler and if necessary a magnifying glass. The units of activity areread from a standard curve with the zones obtained by a dilution seriesfor the synthetic β-defensin peptide (i.e., hBD-3 synthetic peptide).Garcia, J. R., et al., Identification of a novel, multifunctionalbeta-defensin (human beta-defensin 3) with specific antimicrobialactivity. Its interaction with plasma membranes of Xenopus oocytes andthe induction of macrophage chemoattraction. Cell Tissue Res, 2001.306(2): p. 257-64. Antimicrobial potency is measured and compared topublished standards (hBD-3 synthetic peptide or streptomycin). Ideally,the square of the diameter of the inhibition zone is proportional to thelog of the concentration of an antibacterial factor. Frohm, M., et al.,Biochemical and antibacterial analysis of human wound and blister fluid.Eur J Biochem, 1996. 237(1): p. 86-92. This cost effective assay isstandardly used as a measure of antimicrobial activity, however itprovides only semi-quantitative results of antibacterial activity.

A minimum inhibitory concentration (MIC) assay is also performed. Thesmallest amount of conditioned medium from NIKS cells transientlytransfected with each of the β-defensin genes required to inhibit thegrowth of the test organism is determined. In this assay a series ofculture tubes (or wells of a multi-well plate) containing bacterialgrowth medium with varying concentrations of NIKS conditioned medium isinoculated with the test organism. After an incubation period theturbidity is measured and the MIC is determined. Synthetic antimicrobialβ-defensin peptides are used as positive controls. The MIC results arecompared to those previously published by others (i.e., stimulatedconcentration range 15-70 ug/gm tissue or 3.5-16 uM. Harder, J., et al.,Mucoid Pseudomonas aeruginosa, TNF-alpha, and IL-1 beta, but not IL-6,induce human beta-defensin-2 in respiratory epithelia. Am J Respir CellMol Biol, 2000. 22(6): p. 714-21. These relative ranges are onlyintended to provide guidance in an effort to achieve a reasonable pointof reference.

Example 17 Organotypic Culture

This Example describes assays for hBD expression in organotypicallycultured NIKS cells. Stable genetically-modified NIKS clones thatdemonstrate greater than two fold higher expression levels and enhancedantimicrobial activity over endogenous β-defensin gene expression inNIKS monolayer cultures are candidates of further characterizationefforts. These efforts include preparing organotypic cultures to assessin vitro skin tissue for normal tissue morphology. A range of β-defensinexpression levels are examined because expression levels that are toohigh may hinder the ability to obtain normal tissue morphology.

NIKS cell clones that exhibit several different increased β-defensinexpression levels are used to prepare human skin substitute tissuesusing organotypic culturing techniques. See, e.g., U.S. application Ser.No. 10/087,388; 10/087,346; 10/087,641 and PCT Application US 02/06088,all of which are incorporated herein by reference. The organotypiccultures consist of dermal and epidermal compartments. The dermalcompartment is formed by mixing normal human neonatal fibroblasts withType I collagen in Ham's F-12 medium containing 10% fetal calf serum andpenicillin/streptomycin and allowing contraction. The epidermalcompartment is produced by seeding NIKS cells on the contracted collagengel in 25 μA of a mixture of Ham's F-12:DME (3:1, final calciumconcentration 1.88 mM) supplemented with 0.2% FCS, 0.4 μg/mlhydrocortisone, 8.4 ng/ml cholera toxin, 5 μg/ml insulin, 24 μg/mladenine, and 100 units/ml P/S. Cells are allowed to attach 2 hours at37° C., 5% CO₂ before flooding culture chamber with media (day 0). Onday 2 cells are fed with fresh medium. On day 4, cells are lifted to theair/medium interface on the surface of a media-saturated cotton pad,which allows the cultures to be fed from below. Organotypic cultures areincubated at 37° C., 5% CO₂, 75% humidity and are fed fresh medium every2 days. By day 10, the NIKS cells stratify to form the basal, spinous,granular and cornified epidermal layers.

Histological sections of skin substitutes tissues formed by geneticallymodified NIKS cells are compared to cultures prepared from unmodifiedNIKS cells. Tissue sections are stained with hematoxylin and eosin tovisualize the stratified epidermal layers. Cultures are examined fortissue morphology. Only those β-defensin-expressing clones that exhibitnormal tissue organization and histology are used.

The organotypic cultures in the initial expression studies are preparedusing cells expressing individual β-defensin transgenes. However,chimeric organotypic cultures ca be prepared by mixing NIKS cellsoverexpressing different β-defensins to achieve a broader range ofantimicrobial activities. The cells expressing β-defensin transgenes canbe used in conjunction with cells derived from a patient (See, e.g.,U.S. Appl. 2002/0192196) or in conjunction with untransfected NIKS cellsso that potency can be adjusted. This strategy provides furtherflexibility in protein expression profiles in skin tissue.

Example 18 Analysis of Stable hBD mRNA Expression in OrganotypicCultures

This Example describes assays for hBD mRNA. Total cellular RNA isisolated from whole tissue samples. This total RNA is used as a templatefor the subsequent first strand cDNA synthesis (reverse-transcriptase)reaction followed by the polymerase chain reaction (RT-PCR).Amplification products are resolved on an ethidium bromide stainedagarose gel. The anticipated PCR products specific for the transgene DNAand mRNA product is 1.5 Kb and 720 bp respectively.

Example 19 Analysis of hBD Protein Expression in Organotypic Cultures

To monitor changes in β-defensin expression in cultured skin substitutetissue, media underlying the cultures are harvested at various times.When organotypic cultures are 10 days old, they are incubated for 48hours in fresh medium. After 48 hours media is harvested every 12 hoursfor four days and the levels of β-defensin protein in the media isdetermined by ELISA and/or Western Blot analysis. A comparison is madeto endogenous gene expression levels of cultured skin substitute tissuesmade with untransfected NIKS cells. In some experiments, tissue lysatesare generated in order to detect β-defensin protein.

Example 20 Antimicrobial Analysis of Stable β-Defensin Clones inOrganotypic Cultures

Inhibition Zone Assay of Antimicrobial Activity: To determine if humanskin substitute tissue generated from β-defensin-expressing NIKS cellsresults in enhanced bactericidal activity, a modified in vitroinhibition zone assay is utilized. Both the conditioned medium andbiopsy punches from 14, 21, and 28 day old skin substitute tissues areanalyzed for antimicrobial activity. Briefly, thin (1 mm) agarose platesare seeded with a microbe of choice (E. coli, S. aureus, P. aeruginosa,S. pyogenes or C. albicans). The melted agarose (1%) containsLuria-Bertani broth with or without supplemented salt. The testorganism, (˜5×10⁴ log-phase cells/ml) is added just before pouring theplate. To assay for β-defensin activity in conditioned medium from skinsubstitute tissues small wells (3 mm diameter) are punched in the assayplates and loaded with 3 ul of harvested culture medium conditioned forat least 24 hours by human skin substitutes generated with untransfectedNIKS or NIKS clones stably expressing each β-defensin. Alternatively,discs may be loaded with 3 ul of harvested conditioned medium describedabove and placed on a plate containing the seeded microbial lawn. Apositive control sample of a synthetic hBD-3 peptide (2-30 ug/ml) or anantibiotic such as streptomycin (100 ug/ml) will be added to theconditioned medium and assayed, along with a negative control(unconditioned medium sample). To assay the human skin substitutedirectly, four 8 mm punches are collected from each 44 cm² circular skinsubstitute tissue. As described above, each biopsy punch is homogenized(PowerGen Homogenizer), and placed on a plate containing the seededmicrobial lawn. After overnight incubation at 30° C., the inhibitionzones are recorded using a ruler and if necessary a magnifying glass.The units of activity are read from a standard curve with the zonesobtained by a dilution series for the synthetic β-defensin peptide(i.e., hBD-3 synthetic peptide). Antimicrobial potency is measured andcompared to published standards (hBD-3 synthetic peptide orstreptomycin). Ideally, the square of the diameter of the inhibitionzone is proportional to the log of the concentration of an antibacterialfactor. Frohm, M., et al., Biochemical and antibacterial analysis ofhuman wound and blister fluid. Eur J Biochem, 1996. 237(1): p. 86-92.This cost effective assay is standardly used as a measure ofantimicrobial activity, however it will provide only semi-quantitativeresults of antibacterial activity.

Micro-broth Dilution Assay:

A minimum inhibitory concentration (MIC) assay is performed. Thesmallest amount of conditioned medium and biopsy punches from 14, 21,and 28 day old skin substitutes from NIKS cells stably transfected witheach of the β-defensin genes required to inhibit the growth of the testorganism are determined. In this assay, a series of culture tubes (orwells of a multi-well plate) containing bacterial growth medium withvarying concentrations of conditioned medium from skin substitutetissues is inoculated with the test organism. To assay the human skinsubstitute directly, four 8 mm punches are collected from each 44 cm²circular skin substitute tissue. As described above each biopsy punch ishomogenized (PowerGen Homogenizer), and varying concentrations areincubated with the test organism. After an incubation period theturbidity is measured and the MIC is determined. Synthetic antimicrobialβ-defensin peptides are used as positive controls. The MIC results arecompared to those previously published by others (i.e., stimulatedconcentration range 15-70 ug/gm tissue or 3.5-16 uM. These relativeranges are only intended to provide guidance in an effort to achieve areasonable point of reference.

Bacterial Growth Assay:

To evaluate antimicrobial effects of β-defensins on microbes, cellculture supernatants from stable NIKS clones (either monolayer ororganotypic cultures) will be evaluated for the ability to inhibitbacterial growth. Cell culture supernatants will be inoculated withapproximately 4×10⁶ c.f.u. of bacteria, in triplicate, and incubated for1-4 hours at 37 degrees. Cell culture media supernatant collected from anative NIKS cell culture (i.e. non-genetically modified) will serve asan experimental control. NIKS cell culture supernatants spiked withpurified β-defensin peptide titrations will be used as positive controlsfor antimicrobial activity. Immediately following the 1-4 hourincubation period, serial dilutions of each culture condition will beplated on LB/agar plates and incubated at 37 degrees for 18-20 hours.Triplicate plates for each serial dilution are assessed for colonyforming units.

Example 21 Expression of Defensins in NIKS Cells

This example describes elevated β-defensin expression levels intransiently transfected NIKS cell monolayer cultures. Purified DNA fromeach of the Involucrin-β-defensin-Ub-Bsdvectors (FIG. 14) was introducedinto NIKS cells using TransIt-Keratinocyte reagent (Mirus Corporation,Madison, Wis.). Mock transfected (no DNA) or empty vector (noβ-defensin) transfected populations of NIKS cells were also analyzed forendogenous expression levels.

Characterization of Transient β-Defensin Transgene Expression inMonolayer NIKS Cell Cultures

Expression of β-defensin mRNA from the involucrin expression constructswas detected in transiently-transfected NIKS monolayer cell cultures byRT-PCR (FIG. 15). Primers were designed to amplify only the β-defensintransgene transcripts from the involucrin expression vectors and do notdetect endogenous β-defensin expression mRNA. Also, to minimizeamplification from DNA template, DNase treatment was performed on eachof the total mRNA samples prior to the first strand cDNA synthesis(reverse transcriptase) reaction. β-defensin specific expression mRNAproducts (arrowhead) can be distinguished from PCR products amplifiedfrom expression vector DNA in that they lack the rabbit β-globin intronand are therefore 600 bp smaller than products amplified from DNA (seeFIG. 14).

The ability to achieve expression of each β-defensin transgene wasexamined using transient transfections. NIKS keratinocyte monolayercells (1×10⁶ per well) were transfected with theInvolucrin-β-defensin-Ub-Bsd plasmid (10 μg) overnight usingTransIT-keratinocyte reagent (Mirus Corporation, Madison, Wis.). Acontrol mock transfection that contained NIKS cells without the additionof plasmid DNA was also included. One day post transfection, cells werecollected. Total RNA was isolated using Trizol reagent (Invitrogen,Carlsbad, Calif.) and was analyzed by RT-PCR to monitor β-defensin geneexpression from each of the Involucrin-β-defensin-Ub-Bsd constructs.

Results of the RT-PCR analysis of β-defensin gene expression are shownin FIG. 15. PCR primers were designed to amplify β-defensin mRNAexpressed from the transgene construct, but not endogenous hBD mRNA.These primers also amplify DNA from the Involucrin-β-defensin-Ub-Bsdplasmids, but this product can be distinguished from the spliced mRNAproduct because it contains the rabbit β-globin intron and so is 600 bplarger than the spliced product (see FIG. 14). A prominent PCR productcorresponding to spliced β-defensin mRNA (arrowhead) is detected forhBD-1, hBD-2 and hBD-3 (FIG. 15 lanes 1, 5, and 9 respectively). Thisproduct is not seen in control reactions lacking reverse transcriptase(FIG. 15 lanes 3, 7, and 11), demonstrating that it is derived frommRNA. These results also show that each of the hBD expression constructsis expressed in NIKS keratinocyte cell cultures.

Expression of Exogenous β-Defensin Protein in NIKS Cells

Culture medium from cells transiently transfected with each of the threeβ-defensin constructs was assayed for overexpression of protein byimmunoblot analysis using anti-β-defensin antibodies specific for hBD-1,hBD-2 (Santa Cruz Biotechnology, Santa Cruz, Calif.) and hBD-3 (SAGEBioVentures, Carlsbad, Calif.).

Conditioned medium and cell lysates from transiently transfectedmonolayer of NIKS keratinocyte cultures were analyzed separately bySDS-PAGE under denaturing, reducing conditions and the levels of hBD-3protein assessed by immunoblot analysis. Transient transfection of NIKSmonolayer cultures was performed and one day post transfection,monolayer culture supernatants and cell lysates were collected aspreviously described for mRNA expression analysis. A BCA protein assaykit (Pierce, Rockford, Ill.) was used to establish a predeterminedamount of protein to be loaded into each well of a 16% Tricine Novexpre-cast gel (Invitrogen, Carlsbad, Calif.) and then electroblotted ontoa PVDF (0.2 μm pore size) filter. After blocking with 4% skim milk inphosphate-buffered saline for 1 hour, the filter was incubated overnightwith a rabbit polyclonal antibody purified against amino acid residues23-33 of the human β-defensin-3 protein (1:500). The filter was thenincubated with goat anti-rabbit IgG horseradish peroxidase-conjugatedsecondary antibody (1:5000) for 1 hour. Products were detected byincubating blots with enhanced chemiluminescence (ECL) immunoblottingdetection system (Amersham Pharmacia Biotech, Sunnyvale, Calif.) andexposing to film.

The anticipated product size of hBD-3 protein is 5 kDa. However recentstudies have reported that hBD-3 protein exhibits a molecular weight ofapproximately 14 kDa, consistent with the formation of a dimer (Schibli,D. J., et al., J Biol Chem, 2002. 277(10): p. 8279-89). This differencein weight may be in part explained by post-translational modification ofproteins (i.e., glycosylation) or is due to dimers of hBD-3 present as aresult of non-reduced disulfide bonds. Synthetic, control hBD-3 (90 ng)was detected by immunoblot analysis (FIG. 16, lane 1). hBD-3 protein ofthe expected molecular weight (5 kDa or 14 kDa) was not detected inconditioned medium harvested from transfected or mock (untransfected)NIKS (see FIG. 16, lanes 4 & 5). The presence of the high molecularweight band observed in lanes 4 and 5 appears to be dependent on thepresence of serum in the conditioned medium. Only a very faint highmolecular weight band was observed in serum-free conditioned mediumharvested from transfected or mock (untransfected) NIKS keratinocytes.

A 14 kDa protein recognized by the anti-hBD-3 antibody in cell lysatesfrom both transiently transfected and mock transfected NIKS cell lysateswas detected (FIG. 16, lanes 6 and 7). NIKS transiently transfected withthe hBD-3 transgene produce increased levels of hBD-3 protein. Thesecell lysate results indicate that, although hBD-3 protein isover-expressed in transiently-transfected NIKS cells, it remainsassociated with the cells or extracellular matrix and does not appear tobe secreted into the medium. The secretory signals for the granules thatcontain sequestered β-defensin peptide appear to be tightly associatedwith late stages of squamous differentiation (Oren, A., et al., Exp MolPathol, 2003. 74(2): p. 180-2).

Example 22 Antimicrobial Activity of β-Defensin in TransientlyTransfected NIKS Cell Monolayer Cultures

This Example describes antimicrobial activity of defensins in cellculture.

Development of an Antimicrobial Assay Used to Detect Biologicalβ-Defensin Activity

The antimicrobial activity assay employed Escherichia coli andStaphylococcus carnosus and is a modification of the protocol describedby Porter and coworkers (Porter, E. M., et al., Infect Immun, 1997.65(6): p. 2396-401). Briefly, gram-positive or gram-negative bacteriaare grown overnight. The following day the test organisms aresubcultured for 2.5 hr and working dilutions of 10⁴ bacteria/ml forEscherichia coli or 10⁵ bacteria/ml of Staphylococcus carnosus in 10 mMsodium phosphate (pH 7.4)-1% TSB are created. All the reactions mix 50μl of experimental reagent (lysis, supernatants or purified protein)with 50 μl of bacterial suspension. These reactions are then incubatedat 37° C. for 1.5 hr. The reactions are diluted 100-fold in 10 mM sodiumphosphate (pH 7.4)-1% TSB and plated on TSB plates using a spiral plater(Spiral Biotech, Norwood, Mass.). The plates are then incubated for 12to 16 hr at 37° C. Colonies on these plates are counted and the numberof viable bacteria is determined and expressed as colony forming unitsper milliliter (CFU/ml).

Standard Curves for Antimicrobial Activity of Synthetic hBD-1, hBD-2 andhBD-3 Peptides

Standard curves for the antimicrobial activity of hBD-1, hBD-2, andhBD-3 are shown in FIG. 17. Among the hBD proteins, hBD-3 exhibited themost antimicrobial activity with the concentration necessary to kill 50%(LC₅₀) of E. coli at 2.4 μg/ml (FIG. 17 a). Both hBD-2 and hBD-1 wereless potent than hBD-3 against E. coli (FIG. 17 b and c). hBD-2 had anLC₅₀ of 12.2 μg/ml for E. coli and hBD-1 had an LC₅₀ of 102 μg/ml. Thegram-positive bacteria, S. carnosus, appears to be even more sensitiveto hBD-3 with an LC₅₀ of 0.19 μg/ml (FIG. 17 d).

Neither conditioned medium nor cell lysates from monolayer cultures ofNIKS cells transiently transfected with hBD transgenes or controlsexhibited antimicrobial activity in the antimicrobial assay. Endogenousexpression of hBD-2 and hBD-3 is observed only in organotypic culturesof NIKS keratinocytes not monolayer cultures. Therefore, the monolayerculture conditions compromise the ability to use transient expressionexperiments to assay for antimicrobial activities of the hBD-2 and hBD-3proteins. Although hBD-1 is expressed in monolayer and organotypiccultures of NIKS keratinocytes, hBD-1 exhibits the lowest antimicrobialactivity in test organisms. In addition, it is possible that thetransient transfection efficiency of NIKS cells, which is generally20-30%, may not be sufficient to support the hBD levels necessary toexhibit antimicrobial activity. These findings led us to the generationof clones of NIKS keratinocytes stably expressing an hBD transgene andto conducting antimicrobial activity assays using organotypic culturesof these NIKS clones.

Isolation of Stably Transfected NIKS Keratinocytes

Based on the lack of antimicrobial activity observed in the transientlytransfected NIKS keratinocytes, stably-transfected NIKS clonesexpressing hBD transgenes were isolated. The present invention is notlimited to a particular mechanism. Indeed, an understanding of themechanism is not necessary to practice the present invention.Nonetheless, it is contemplated that higher levels of hBD expressionwould be achieved in clones stably-transfected with the hBD transgenes.In addition, it was observed that both endogenous hBD mRNA and proteinlevels were enhanced by organotypic culture NIKS keratinocytes and thatstratification and/or late stage differentiation events associated withthe development of barrier function may be necessary for hBD processingor secretion. Transiently transfected NIKS keratinocytes cannot beassayed following organotypic culture because full stratification andbarrier function requires at least 11 days to develop and transientexpression of hBDs would be exhausted.

Stable clones of NIKS keratinocytes expressing hBD-3 were firstgenerated. hBD-3 was selected because it demonstrates the most potencyagainst the two test organisms and exhibits antimicrobial activityagainst both gram positive and gram negative bacteria. Multipleindependent clones expressing the hBD-3 transgene were obtained bytransfecting NIKS cells and selecting stably-transfected cells usinggrowth medium containing blasticidin (2.5 μg/ml). Elevated β-defensinmRNA expression was verified using RT-PCR analysis of total RNA isolatedfrom each NIKS clonal cell line and served as an initial screen forrelative expression levels between different clones.

NIKS clones stably expressing the hBD-3 transgene have been isolated andscreened. To quantify relative β-defensin expression levels instably-transfected NIKS Involucrin-Defensin-3-Ub-Bsd clones, totalcellular RNA was isolated from blasticidin-resistant clones. RT-PCRanalysis was performed on all blasticidin-resistant NIKS clonestransfected with the Involucrin-Defensin-3-Ub-Bsd expression construct.

Conditioned Medium from Organotypic Cultures of NIKS™ Clones StablyExpressing the hBD-3 Transgene Exhibit Enhanced Antimicrobial Activity

The antimicrobial activity of conditioned medium harvested fromorganotypic cultures of the stably-transfected NIKS keratinocyte cloneexpressing the highest level of hBD-3 mRNA was assayed using the methoddescribed above. FIG. 18 shows that 70% of the E. coli and up to 52% ofthe S. carnosus bacteria were killed following exposure to conditionedmedium from organotypic cultures of NIKS keratinocytes stably expressingthe hBD-3 transgene when compared to conditioned medium harvested fromorganotypic cultures generated from untransfected NIKS keratinocytes.Conditioned medium from control NIKS organotypic cultures exhibitdetectable, but low levels of antimicrobial activity, consistent with alink between squamous differentiation and endogenous hBD-3 expression(Abiko, Y., et al., J Dermatol Sci, 2003. 31(3): p. 225-8.).

Example 23 Defensin Mutants

This example describes site-directed mutagenesis of hBD3. Five (5) ofsix Cys were mutated to Ala (i.e., Cys₄₀, Cys₄₅, Cys₅₅, Cys₆₂, Cys₆₃).In another mutant, Gly₃₈ is mutated to Ala₃₈).

Site-Directed Mutagenesis—

A commercially available kit, QUIKCHANGE Multi Site-Directed Mutagenesiskit (Stratagene, LaJolla, Calif.) was used to create amino acidsubstitutions in the native hBD-3 polypeptide. The hBD-3 cDNA TopoTA DNAvector was used as the parental DNA template for the site-directedmutagenesis reactions using the manufacturer specifications. Briefly, athermocycling reaction included—double stranded DNA template, two ormore synthetic phosphorylated oligonucleotide primers that contain thedesired mutation(s), enzyme blend containing PfuTurbo DNA polymerase.First the mutagenic primers are annealed to the denatured DNA template.PfuTurbo DNA polymerase was used to extend the mutagenic primer(s)generating double stranded DNA molecules with one strand bearing thewanted mutation(s). In Step 2, the thermocycling reaction products weretreated with Dpn I restriction endonuclease. The Dpn I endonuclease isspecific for methylated and hemimethylated DNA and is used to digestparental DNA template. DNA isolated from almost all E. coli strains isdam methylated and therefore susceptible to this digestion. In Step 3,the reaction mixture, enriched for mutated single stranded DNA istransformed into ultracompetent cells (dam⁺), where the mutant closedcircle ss-DNA is converted to duplex form in vivo. Double strandedplasmid DNA is prepared from the transformants and clones are identifiedthat contain the wanted mutation(s).

Synthetic Phosphorylated Oligonucleotide Primers—

The mutated codon sequence is underlined.

1) Gly₃₈→Ala Mutation Oligonucleotide Sequence

(SEQ ID NO: 115) (ST262) 5′-Phos-GCA GAG TCA GAG GCG CCC GGT GTG CTGTGC TCA GC-3′2) Cys_((40,45,55,62,63))→Ala Mutation Oligonucleotide Sequences

(SEQ ID NO: 116) (ST258) 5′-Phos-CCTCCTTTGGAAGGGCGCTGAGCACAGCAGCCCGGCCGCC-3′ (SEQ ID NO: 117)(ST259) 5′-Phos-CTTTCTTCGGGC GGCTTTTCGGCCACGCGTCGA GGCCTTGCCGATC-3′Final Mutant Amino Acid Sequences—

The site-directed substitutions are highlighted.

1) Amino Acid Sequence (Gly38→Ala)

(SEQ ID NO: 118) MRIHYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGARCAVLSCLPKEEQIGKCSTRGRKCCRRKK2) Amino Acid Sequence (5Cys→Ala)

(SEQ ID NO: 119) MRIHYLLFALLFLFLVPVPGHGGIINTLQKYYCRVRGGRAAVLSALPKEEQIGKASTRGRKAARRKKExpression Vector Constructs—

Involucrin promoter hBD-3 mutant cDNA Globin poly(A)

Electroporation Transfection Method—

Early passage NIKS cells were harvested at @approximately 50-70%confluence. Cells were pelleted and the pellet resuspended (1×10⁶-3×10⁶cells/800 ul) in F-12/DME (5:1).

800 ul of NIKS cell suspension was placed in 0.4 cm electroporationcuvette, DNA was added (10-30 ug, linear or supercoiled), placed incuvette holder of the GenePulser and started. All steps were done atroom temperature; the cells were not placed on ice at any time duringthis procedure. The actual voltage and capacitance values were recorded

Electroporated NIKS cells were removed from the cuvette and diluted into25-50 mls of fresh NIKS medium, mixed well by pipetting, and plated(5-10 mls) per p150 containing blasticidin resistant feeders (usingeither 5 or 10 p150's per transfection reaction).

In the next 24-48 hours, the medium is replaced on the p150's withblasticidin containing medium (2.5 ug/ml blasticidin).

BioRad GenePulser Electroporation Settings:

Exponential Pulse Program

270 volts

950 uF

∞ ohms

0.4 cm cuvette

Selection—

NIKS keratinocyte clones were cocultured in the presence of blasticidinresistance feeder cells and selected for growth in presence of NIKSmedium containing 2.5 ug/ml blasticidin. Only those colonies thatcontinued to grow in the presence of blasticidin selection for durationof selection (a minimum of 18 days) were isolated and expanded forfurther characterization.

Clone Isolation—

A traditional “Ring cloning” method is to isolate blasticidin resistantcolonies. The clones are first picked onto a feeder layer in individualplates (p60) and allowed to grow until they are between 80-90%confluent. The clones are then passed and re-plated to two individualtissue culture plates (p60 and p100). The p100 contains mouse fibroblastfeeder cells and the p60 does not. When these cultures reach 80-90%confluence, the p60 cultures are harvested for expression analysis andthe p100 cultures are used for the subsequent expansion phase.

Characterization of Stably-transfected NIKS keratinocytes—

Stable NIKS keratinocyte colonies that survived the selection schemetherefore are presumed to contain the Involucrin hBD-3 expressionconstruct. To confirm the presence of the hBD-3 transgene, RNA wasisolated from each clone and cDNA products were generated using reversetranscription (RT). The RT products were then used as templates insubsequent PCR reactions. This PCR screen was designed to reconcileproducts derived from transgene cDNA from that of potential endogenoushBD-3 DNA products. Multiple clones were obtained using the hBD-3constructs (Gly38→Ala substitution and 5 Cys→Ala substitution) andassociated selection scheme.

Expansion—

The results of expression analysis obtained from the p60 culturesdictate which clones were expanded for further characterization. Thep100 plates from cultures identified as having positive expression weregrown to approximately 90% confluence then harvested and frozen at −80°C. in media containing 10% glycerol.

Results are shown below.

Experiment # (Clones picked/Positive) Exp 1-Gly→ Ala* 10/10 Exp 2-Gly→Ala 14/16 Exp 3-Gly→ Ala 11/11 Cys→ Ala**  0/2 Exp 4-Cys→ Ala  2/2 Exp5-Cys→ Ala  4/4 Exp 6-Cys→ Ala  3/3 (two more yet to be screened) Exp7-Cys→ Ala 25/26 (five clones yet to be screened) *Mutant construct(Gly→ Ala)-hBD-3 amino acid substation **Mutant construct (Cys→Ala)-hBD-3 amino acid substation five of six cysteines to alanines.

Example 24 Design and Construction of hCAP18 Expression Vectors

This example describes the design and construction of human cathelicidin(hCAP18) mammalian expression vectors.

The human cathelicidin (hCAP18) cDNA was cloned from a commerciallyavailable human cDNA library. PCR products were amplified sequenced toconfirm the genetic identity. This hCAP18 cDNA sequence was confirmed tobe identical to that sequence deposited in Genbank for hCAP18.

Two hCAP18 mammalian expression vectors were generated. The first vectorcontains the tissue specific keratin-14 promoter, and the second vectorutilizes the involucrin promoter as an alternative promoter strategy. Alinear map and diagnostic digests of the hCAP18 mammalian expressionvectors are shown in FIG. 19. The diagnostic restriction enzyme digestsdemonstrate correct banding patterns of the appropriate sizes. Takentogether these results account for the overall integrity of themammalian expression constructs. Sequencing across all of the cloningjunctions on both final assembled constructs (K14 hCAP18 and involucrinhCAP18) was also performed to verify the sequence integrity of eachexpression vector.Expression of hCAP18 from Expression Constructs

RT-PCR analysis was conducted to verify overexpressed levels of hCAP18from both expression constructs (FIG. 20). Reverse transcriptasereactions were performed on monolayer NIKS cell cultures transientlytransfected with each of the human cathelicidin expression vectors ormock transfected. The anticipated PCR product size of 0.6 kbcorresponding to hCAP18 is shown in transfected cells and as expectedthis hCAP18 product is not seen in RNA from Mock transfected monolayerNIKS cell cultures. An additional set of PCR primers specific for anendogenous house keeping gene (GAPDH) was used on the RT reactions tocontrol for RNA integrity and first strand cDNA synthesis reactions.

Example 25 hCAP18/LL-37 Antimicrobial Activity

This example describes the development of an assay to detect LL-37antimicrobial activity. In developing this assay a standard kill curvewas produced using a commercially available LL-37 peptide (PhoenixPharmaceuticals, Belmont, Calif.). The assay is a modification of theantimicrobial assay developed to assess biological activity of otherantimicrobial peptides described above. A standard curve for theantimicrobial activity of LL-37 was determined for gram-positivebacteria, S. carnosus using this synthetic peptide. Results indicatedthat LL-37 exhibited potent antimicrobial activity with a concentrationnecessary to kill 50% (LC₅₀) of the S. carnosus at 0.9 ug/ml.

Example 26 Electroporation of Cells

This Example describes the use of electroporation to introduce nucleicacids into keratinocytes. This Example further describes the use ofelectroporation to select for pluripotent and multipotent cells in apopulation.

Protocol:

Harvest early passage NIKS cells @approximately 50-70% confluence.Pellet cells and resuspend NIKS cell pellet (2×10⁶ cells/800 ul) inF-12/DME (5:1). This same protocol electroporating 1×10⁶ NIKS cells in800 ul with the same success.

Place 800 ul of NIKS cell suspension in 0.4 cm electroporation cuvette,add DNA (10-30 ug, linear or supercoiled) place in cuvette holder of theGenePulser and push button. All steps are done at room temperature; thecells are not placed on ice at any time during this procedure. Recordactual voltage and capacitance values (these values are indicative ofreproducible electroporation experimental conditions and may be usefulfor future reference).

Electroporated NIKS cells are removed from the cuvette and diluted into25-50 mls of fresh NIKS medium, cells are mixed well by pipetting, andplated (5-10 mls) per p150 containing blasticidin resistant feeders(using either 5 or 10 p150's per transfection reaction).

The following day replace medium on the p150's with blasticidincontaining medium (2.5 ug/ml blasticidin). Clonal selection of NIKSkeratinocytes is typically carried out for 18-20 days in blasticidinmedia (with fresh medium changes every other day).

The traditional electroporation conditions for mammalian cells asprovided by the manufacturer (BioRad) are described below. Theseconditions need to be optimized; they are equipment specific and celltype specific.

Electroporation Medium recommended to be minimal or TE at 0.5-0.8 mls.

Cell Density (single cell suspension) 6-8 × 10⁶ Volume of Cells 0.4-0.8mls DNA 20-200 ugGene Pulser (BioRad) Technical Services Recommended Ranges Using anExponential Protocol.

Gene Pulser Settings

Voltage (V) 200-350 Capacitance (μF) 500-1000 Resistance (Ω) ∞ Cuvette(mm) 0.4

Experiments conducted resulted in the following optimized protocol forelectroporation:

Cell Density (single cell suspension) 1-2 × 10⁶ Volume of Cells*  0.8mls DNA** 10-20 ug *F-12/DME minimal medium (50 mls:10 mls) **Linear orsupercoiled DNA (Qiagen Maxiprep DNA purification) All steps performedat ambient temperatureGene Pulser Settings

Voltage (V) 270 Capacitance (μF) 950 Resistance (Ω) ∞ Cuvette (mm) 0.4

The above protocol was used to select for cells in a populations ofcells that have stem-cell-like keratinocyte populations. In someembodiments, a drug selection cassette was electroporated. In otherembodiments, the cells are electroporated in the absence of anyexogenous nucleic acids. The results are described below.

-   I. Clonally selected cell population observations (Drug selection    cassette containing DNA electroporated and cell populations under    drug selection for >18 days):    -   1) Selected for keratinocytes having holoclone or meroclone cell        morphology-colony morphology of tightly packed, uniform cells,        smooth colony edges, overall round colony morphology.    -   2) Selected for cells with stem-cell-like properties    -   3) Selected for cells that exhibit extended proliferative        capacity—in creation of stable cell lines, these colonies are        typically the only surviving colonies after >18 days under drug        selection pressure.    -   4) Selected for cells with enhanced pluripotency or        multipotency.    -   5) Colonies without holoclone or meroclone morphology remain        smaller and tend to stop growing. These colonies do not share        the same characteristics as does the small tightly packed        uniform cells within each large colony. These colonies die-off        and most detach from the plate during the selection process.-   II. Electroporation population observations (Exposed to    electroporation conditions w/o DNA and not placed under selection):    -   1) Selected for keratinocytes having holoclone or meroclone cell        morphology-colony morphology of tightly packed, uniform cells,        smooth colony edges, overall round colony morphology.    -   2) Selected for cells with stem-cell-like properties    -   3) Selected for cells that exhibit extended proliferative        capacity—these colonies are typically the larger surviving        colonies    -   4) Selected for cells with enhanced pluripotency or        multipotency.    -   5) Colonies without holoclone or meroclone morphology remain        smaller and tend to stop growing. These colonies do not share        the same characteristics as does the small tightly packed        uniform cells within each large colony.

The results of this experiment demonstrated that populations of cell canbe electroporated with or without exogenous nucleic acid and cells withthe above described properties are selected for. In addition, Transgeneexpression from NIK stable clones obtained using the electroporationmethod of selection have higher expression levels when compared to thoseclones obtained using the Trans-IT keratinocyte (Minis) transfectionmethod as demonstrated with semi-quantitative RT-PCR analysis.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention that are obvious to those skilled inmolecular biology, biochemistry, or related fields are intended to bewithin the scope of the following claims.

The invention claimed is:
 1. An organotypic human skin equivalentcomprising stratified squamous epithelia produced from Near-diploidImmortalized Keratinocytes (NIKS) cells stably transfected with a vectorcomprising a nucleotide sequence encoding cathelicidin polypeptide andwherein the nucleotide sequence is operably linked to a promotersequence selected from the group consisting of the involucrin promoterand the keratin-14 promoter, wherein said skin equivalent exhibitsantimicrobial activity.
 2. The organotypic human skin equivalent ofclaim 1, wherein said human skin equivalent further compriseskeratinocytes from a patient.
 3. The organotypic human skin equivalentof claim 1, further comprising a dermal equivalent.
 4. The organotypichuman skin equivalent of claim 1, further comprising Near-diploidImmortalized Keratinocytes (NIKS) cells that do not express exogenouscathelicidin.
 5. The organotypic human skin equivalent of claim 1,further comprising keratinocytes expressing at least one additionalantimicrobial polypeptide.